Whole-slide imaging methods for microscopy

Abstract

A method is provided for operating a microscope scanner (1) comprising a line scanner (3). Pre-scan data for a target (6) comprising a sample is generated, the generating the pre-scan data comprising obtaining a pre-scan image of the target (6) from the pre-scan and identifying one or more sample-containing regions (24) and sample-absent regions (26) of the target (6) from the pre-scan image. An imaging scan of a scan region of the target (6) comprising the one or more sample-containing regions (24) and one or more sample-absent regions (26) is then performed. The scanning speed of the line scanner (3) is adjusted along an imaging scan path based on the pre-scan data such that the target (6) is imaged at a higher scanning speed within the one or more sample-absent regions (26) than within the one or more sample-containing regions (24).

Description

[Technical field]

[0001] The present invention relates to a method for operating a microscope scanner. [Background technology]

[0002] Whole slide scanners scan pathological or histopathological samples mounted on microscope slides under bright field observation conditions. The term whole slide scanner is used because it is intended that all of the tissue sample on the slide is scanned. A pre-scan of the whole slide is typically performed to generate a low resolution "pre-scan image". This image is then analyzed to identify one or more areas of interest ("AOI") on the target where the sample is suspected to be present, and these areas are selectively imaged during a subsequent high resolution imaging scan. Typically, the imaging scan will be performed using a scanning resolution of 2,000-4,000 lpmm (lines per millimeter, each image acquired by a line scanner corresponds to an individual line) or equivalently 51,000-101,000 dpi (dots per inch). The pre-scan will instead be performed at a much lower resolution, typically between 12-61 lpmm (300-1500 dpi). The pre-scan can therefore be performed much faster (at least an order of magnitude faster) than the imaging scan. Target areas outside the AOI are therefore excluded from the imaging scan to reduce the overall scan time.

[0003] When tissue is comparable to or smaller than the pixel size in the pre-scan, it does not provide good contrast in the pre-scan image that is acquired. As a result, analysis of the pre-scan image to detect the presence of tissue may fail to detect the tissue. This would result in tissue not being scanned in the high resolution image. To alleviate this problem, pre-scan images of increasingly higher resolution can be generated to detect ever smaller tissue areas. However, as the resolution of the pre-scan image increases, it takes longer to perform the pre-scan and longer to analyze the pre-scan image to find tissue regions.

[0004] The pre-scan image typically has a large depth of field compared to the thickness of the tissue. This has the advantage that no focusing step is required to perform the tissue scan, but has the disadvantage that defects such as dust and scratches on the surface of the slide are also focused. This forces the AOI detection algorithm to distinguish between surface defects and tissue. This discrimination between tissue and defects is not trivial, and a compromise must be found between the time to analyze, how much tissue is overlooked by the AOI detection algorithm, and how much defects are falsely identified as tissue by the AOI detection algorithm. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] US 9116035 B2 [Non-patent literature]

[0006] [Non-Patent Document 1] "Numerical Recipes in C: the Art of Scientific Computing", Cambridge University Press, 1992, Chapter 3 Summary of the Invention [Problem to be solved by the invention]

[0007] It is desirable to ensure that the entire sample is imaged during an imaging scan without significantly compromising the speed or efficiency of the scanning process, and it is in the context of solving this problem that the present invention is set out. [Means for solving the problem]

[0008] A first aspect of the present invention provides a method of operating a microscope scanner comprising a line scanner, the method comprising the steps of generating pre-scan data for a target comprising a sample, the generating step comprising obtaining a pre-scan image of the target from the pre-scan and identifying from the pre-scan image one or more sample containing regions corresponding to locations on the target where a sample of the target is expected to be present and one or more sample absent regions corresponding to locations on the target where a sample is expected to be absent; performing an imaging scan of a scan area of ​​the target including the one or more sample containing regions and the one or more sample absent regions, the performing step comprising moving the line scanner relative to the target along an imaging scan path and obtaining images of the target at each of a plurality of locations along the imaging scan path using the line scanner, and adjusting a scanning speed of the line scanner along the imaging scan path based on the pre-scan data such that the target is imaged at a higher scanning speed within the one or more sample absent regions than within the one or more sample containing regions.

[0009] The pre-scan data is analyzed to determine areas that contain a sample (typically tissue) and areas where the sample is believed to be absent. However, since errors may arise from the analysis of the pre-scan data and it is desirable to ensure that all of the sample is imaged, rather than performing the imaging scan only in areas detected as containing a sample, the "sample absent regions" are also imaged as a precaution. Optionally, this may be extended to image the entire target during the imaging scan. The scanning speed is increased in one or more sample absent regions during the imaging scan, typically resulting in a corresponding reduction in image resolution in these regions. This allows the sample detection algorithm to make a compromise between tissue detection and defect detection, with fewer defects being erroneously identified as tissue. Furthermore, even if the sample is imaged at a lower resolution in the nominal "sample absent regions" than in the "sample containing regions", the data generated is typically more useful than if the sample absent regions were not imaged at all. The increased scanning speed in the sample absent regions ensures that any additional time penalty incurred is minimal.

[0010] The step of performing the imaging scan preferably further comprises monitoring one or more image parameters from images of the target acquired along the imaging scan path, and further adjusting the scanning speed of the line scanner in one or more sample-free regions in response to changes in such one or more image parameters. By doing so, the scanner can react to the (unexpected) presence of a sample in the "sample-free region". When a sample enters the field of view of the line scanner, it will lead to a corresponding change in the monitored one or more image parameters. The scanning speed is adjusted in response to this change, thereby allowing the sample to be imaged with high resolution during the imaging scan regardless of its location on the target. Thus, a higher percentage of the sample on the target can be imaged, which improves the overall usefulness of the scanned image to the microscope user. With this technique, the scanning speed can be adjusted on the fly in response to the monitored image parameters (typically related to the amount of detail in the image), so that the spatial resolution of the scan is not predetermined by the scanner at the time the imaging scan begins.

[0011] One or more image parameters are typically monitored to detect the presence of a sample in one or more sample-free regions, and the scan speed is adjusted so that locations on the target where a sample is detected are imaged at a slower scan speed than locations on the target where a sample is not detected. This allows a compromise to be satisfied between imaging the entire target at high resolution (which is desirable but time consuming to capture the entire sample) and imaging only those areas identified from pre-scan images as containing a sample. Furthermore, if a sample is detected outside the sample-containing regions, the scanner responds to ensure that the sample is imaged at a relatively slower speed and therefore at high resolution, regardless of where it is present on the target.

[0012] Further adjusting the scan speed based on the preliminary scan data preferably comprises imaging the target in one or more sample-free regions at a first target speed and imaging the target in one or more sample-containing regions at a second target speed, the first target speed being higher than the second target speed. Further adjusting the scan speed of the line scanner in one or more sample-free regions thus preferably comprises reducing the scan speed from the first target speed in response to a change in one or more image parameters indicative of detection of a sample. The sample-free regions may thus be imaged at the first target speed only if the monitored one or more image parameters indicate a first criterion (typically the absence of a sample in the acquired image). If the one or more image parameters instead indicate a second criterion (typically the presence of a sample in the acquired image), the scan speed is reduced from the first scan speed to or towards the second target speed. Optionally, the sample-containing region may be imaged at a second target velocity if one or more monitored image parameters indicate either the first or second criterion.

[0013] During the imaging scan, the movement speed can be reduced when the pre-scan data indicates that the edge of the sample is approaching so that the sample is imaged with high resolution throughout. To avoid sudden deceleration, this can be done over the area before the edge of the tissue is reached. The imaging scan can therefore further comprise reducing the speed from a first target speed to or towards a second target speed before the line scanner transitions from acquiring images of the sample-free area to acquiring images of the sample-containing area. The transition between the speeds is advantageously adjusted to occur in the area of ​​the target where the sample is absent. The line scanner can then move at a constant speed over the sample-containing area during the imaging scan. This ensures that the sample-containing area is imaged with uniformly high resolution throughout. However, the imaging scan can further comprise increasing the scanning speed in one or more sample-containing areas in response to a change in one or more image parameters indicative of the absence of a sample in the acquired image. For example, this may occur when an error occurs in identifying the sample-containing area from the pre-scan data. If one or more image parameters monitored during the imaging scan indicate that the region being imaged does not contain a sample, the scan speed can be increased to reduce the total scan time.

[0014] The step of adjusting the scanning speed typically comprises reducing the scanning speed of the line scanner in response to one or more image parameters varying in a first predetermined manner relative to a threshold value. For example, the one or more image parameters may vary in a first predetermined manner relative to the threshold value in response to an image of the sample being acquired in a sample-free region. Similarly, the step of further adjusting the scanning speed typically comprises increasing the scanning speed of the line scanner in response to one or more image parameters varying in a second predetermined manner relative to the threshold value. The threshold value is preferably predetermined and stored in memory, for example following calibration of the scanner. The one or more image parameters may comprise image brightness, and the scanning speed of the line scanner is reduced in response to the image brightness decreasing. Similarly, the one or more image parameters may comprise a focus merit value, and the scanning speed of the line scanner is reduced in response to the focus merit value increasing. Typically, the focus merit value is a numerical measure of the complexity in an image, the higher the value, the more detail in the image. A more focused image has a corresponding higher merit value. The imaging scan preferably comprises the steps of calculating a focus merit value at each location along the image scan path, and adjusting the focus height of a line scanner along the image scan path based on the focus merit value.

[0015] Images are typically acquired at a constant rate in time throughout the scan area during an imaging scan. Thus, image capture is not linked to any particular measurement, but instead can be independently controlled by a timer function. Furthermore, the line scanner is typically moved continuously relative to the target along the image scan path during the imaging scan. Thus, the width of each pixel (and therefore the overall resolution of the image in a particular area of ​​the target) will typically vary according to the speed at which the line scanner moves relative to the target when the image is captured. Thus, a low-resolution image will correspond to an image in which the line scanner has moved over a larger portion of the target between each successive image capture than the high-resolution image. Typically, the spatial resolution of an imaging scan will not be constant across the image scan path, but will depend on the scan speed for a given location on the target.

[0016] It is particularly desirable that the pre-scan be fast to perform. Focus data on the target may not be available when the pre-scan is performed, and therefore a different camera having a greater depth of focus than that of the line scanner may typically be used for the pre-scan. Preferably, the depth of focus for the pre-scan is greater than 500 micrometers (optionally at least 1 millimeter), whereas the depth of focus for each image acquired along the imaging path may be less than 3 micrometers (typically 1 micrometer). This typically ensures that the entire sample is located within the depth of focus for the pre-scan camera. Typically, the pre-scan image is acquired from a single pass of the pre-scan camera along a pre-scan imaging path, which may be linear or in a raster format. Typically, the pre-scan image is acquired at a constant spatial resolution across the pre-scan imaging path.

[0017] Preferably, generating pre-scan data further comprises performing image analysis of the pre-scan image to generate a probability map indicative of the likelihood that a sample is present at each location on the target, and one or more sample-containing regions and one or more sample-absent regions are selected from the probability map. Adjusting the scan speed of the line scanner along the image scan path based on the pre-scan data may then comprise adjusting the scan speed according to the likelihood that a sample is present at a given location on the target as determined from the probability map. The one or more sample-absent regions may correspond to locations from the probability map that are unlikely to comprise a sample, and the one or more sample-containing regions may correspond to locations from the probability map that are likely to comprise a sample.

[0018] In addition to the sample-absent and sample-containing regions, one or more other regions of the target may be identified where the probability map is unable to conclude as to whether a sample is present in the region. For example, generating pre-scan data may further comprise identifying one or more uncertainty regions on the target from the probability map where the likelihood of having a sample is between the likelihood of a sample-absent region and the likelihood of a sample-containing region. A different scan speed may be set for the region than within the sample-absent and sample-containing regions. For example, adjusting the scan speed based on the pre-scan data may comprise imaging the target in the one or more sample-absent regions at a first target speed, imaging the target in the one or more sample-containing regions at a second target speed, and imaging the target in the one or more uncertainty regions at a third target speed, the third target speed being higher than the second target speed and lower than the first target speed. When one or more image parameters being monitored indicate the presence of a sample within the field of view of the line scanner at any point across any of these three regions, the scan speed can be set to a target speed for the sample-containing region (in this case the second target speed).

[0019] Typically, a high resolution image of the target is generated from a combination of images acquired during an imaging scan. Preferably, generating the high resolution image comprises using image interpolation to increase the resolution of images acquired at higher scanning rates to correspond to the resolution of images acquired at lower scanning rates. Thus, typically, the scanning rates at which each image is captured during an imaging scan are stored in memory. Generating the high resolution image may comprise adjusting an interpolation ratio to maintain a constant resolution in the high resolution image.

[0020] The microscope scanner is preferably a bright field microscope, and the imaging scan is preferably performed using a transmission mode for the microscope scanner. The microscope scanner can be configured to operate in a reflection mode. Either or both modes can be used as part of the pre-scan. For example, the transmission mode can be advantageous for identifying sample-containing and sample-absent regions, whereas the reflection mode can be used to identify regions of an opaque target, such as where a label can be fixed to the target. Opaque regions do not need to be scanned during the imaging scan, and thus the scan region is preferably selected from the pre-scan image to substantially cover the entire target excluding any opaque target regions.

[0021] A second aspect of the present invention provides a computer program product comprising instructions which, when executed with a microscope scanner comprising a line scanner and a target comprising a sample, cause the microscope scanner to perform the method of the first aspect. The second aspect shares similar features and advantages as discussed with respect to the first aspect.

[0022] The microscope scanner is preferably a virtual microscope, and the computer program product typically corresponds to a non-transitory computer readable medium such as a hard drive. The target can take several forms, but preferably comprises a biological tissue sample. Preferably, the target does not exhibit substantial changes in surface height, typically for the purposes of a pre-scan with a large depth of focus, and is therefore essentially flat. For example, the target could be a stained tissue extract that has been sliced ​​and held between flat glass slides. The focus of the line scanner can be adjusted during the high resolution imaging scan, since the surface height of the target can change by an amount greater than the depth of focus used in the imaging scan.

[0023] Embodiments of the present invention will now be discussed with reference to the following figures. [Brief description of the drawings]

[0024] [Figure 1] FIG. 1 is a schematic diagram of a microscope scanner to be operated according to a first embodiment of the present invention; [Diagram 2] 3 is a flow chart showing a process according to a first embodiment of the present invention. [Diagram 3] FIG. 2 is a diagram of a pre-scan image acquired in accordance with a first embodiment of the present invention. [Figure 4] FIG. 2 is a diagram of a pre-scan image acquired in accordance with a first embodiment of the present invention showing a scanning area. [Diagram 5] FIG. 2 is a pre-scan image acquired in accordance with a first embodiment of the present invention showing sample-absent and sample-containing regions. [Figure 6] 4 is a graph showing the change in scan speed when going from a sample-free region to a sample-containing region according to a first embodiment of the present invention. [Figure 7] 4 is a graph showing the change in scan speed when going from a sample-containing region to a sample-absent region according to a first embodiment of the present invention. [Figure 8]4 is a graph showing the change in scan speed when detecting the presence of a sample inside a sample-free region according to a first embodiment of the present invention. [Figure 9] 4 is a graph illustrating the interpolation of data when generating a high resolution image of a target according to a first embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] An example of a brightfield microscope scanner 1 is shown in FIG. 1. The brightfield microscope scanner 1 comprises a scan head 2 comprising a line scanner 3 in the form of a linear photodetector array. The scan head 2 further comprises an adjustable focusing system 4 and a pre-scan camera 12. A platen 5 is provided on which a target 6 in the form of a pathology slide comprising a tissue sample is placed. A drive mechanism 7 mounts the scan head 2 on a track 8 so that the scan head 2 can be moved relative to the target 6 as shown by the arrow 9. The track 8 can make it possible to displace the scan head 2 laterally to image adjacent scan widths. The microscope scanner 1 is controlled by means of an electronic controller in the form of a computer system 10. FIG. 1 further provides coordinate axes for reference in the following examples. The ordinate z-axis is aligned with the optical axis of the scan head 2 and with the focal height of the target 6, whereas the abscissa x-axis represents the scanning direction (parallel to the arrow 9 in FIG. 1). The surface of the target 6 is aligned in the xy-plane and the lens of the adjustable focusing system 4 is movable along the z-axis. In an alternative example, the scan head 2 remains stationary and the target 6 is moved in the xy plane to achieve a relative motion between the scan head 2 and the target 6 during the scanning process. The pre-scan camera 12 can form a separate unit from the scan head 2 and can be operated independently of the scan head 2. The pre-scan camera 12 can comprise a line scanner, but the choice is not particularly important as long as the pre-scan camera 12 can be used to acquire a two-dimensional pre-scan image of the entire slide. In contrast, for the imaging scan following the pre-scan, a line scanner 3 must be used. As will be discussed later, neither TDI sensors nor 2D sensors operating in a two-dimensional imaging mode are suitable for imaging scans due to the need to change the spatial resolution during the imaging scan. An example of a suitable microscope scanner 1 is the VENTANA DP200 offered by Roche Diagnostics.

[0026] The operation of the microscope scanner 1 in carrying out a method according to a first embodiment of the invention will now be described with reference to Figures 1 to 9. The method starts at step 100 (Figure 2), where a pre-scan is performed to generate pre-scan data comprising a pre-scan image of the target 6. The scan head 2 passes along the trajectory 8 in one pass along the pre-scan imaging path while acquiring an image of the target 6 with the pre-scan camera 12. The pre-scan camera 12 is configured to acquire an image of the target with a focal depth of 1 mm, which is significantly larger than the thickness of the sample (typically between 2 μm and 10 μm). Thus, the focal height of the pre-scan camera 12 is not adjusted during the pre-scan, and the pre-scan image is generated quickly with a relatively low resolution, for example 1200 dpi (47 lines per mm). The pre-scan image provides an overview of the contour of the sample on the target 6, from which one or more areas of interest AOI can be selected for imaging during a first imaging scan.

[0027] An example of a pre-scan image is illustrated by Fig. 3. On the right side, an opaque area of ​​the target 6 is discernible, where a label 22 is fixed to the slide. As known in the art, this area is identified by operating the microscope scanner 1 in transmission and reflection modes during the pre-scan and comparing the images acquired. The remaining (optically transparent) part of the target 6 is selected by the computer system 10 as the scan area 20 where the imaging scan is to be performed. This scan area 20 is identified by the herringbone pattern superimposed on the pre-scan image in Fig. 4.

[0028] The pre-scan image is processed using conventional software in step 101 such that each area within which a sample is visible is identified as an AOI, also referred to as a "sample-containing area." More specifically, an automated image analysis of the pre-scan image is used to generate a probability map indicating where the sample is expected to be present on the target, and one or more AOIs are selected from the probability map. Typically, the boundaries of the AOI may be selected to extend slightly outside the outer edge of the sample (as identified from the pre-scan image) so that the sample is contained entirely within the AOI. The pre-scan image with the one or more AOIs demarcated may then be displayed to a user using computer system 10. Optionally, the user may then manually adjust the boundaries of the one or more AOIs as needed using the computer system. For illustrative purposes, FIG. 5 shows the pre-scan images of FIGS. 3 and 4, but without the herringbone pattern superimposed over the sample-containing areas 24 identified from the pre-scan image. The remainder of the scan area 20 (outside the sample-containing area 24 ) through which the herringbone pattern extends is referred to herein as the “sample-free area” 26 .

[0029] The imaging scan proceeds at a fairly slow rate compared to the pre-scan, and therefore it is desirable to image only the areas on the target 6 where the sample is present during the imaging scan. Thus, typically, the imaging scan will be performed only over one or more AOIs identified from the pre-scan data. However, the resolution of the pre-scan image is relatively low, and errors may occur in the AOI detection process. Thus, parts of the sample may be inadvertently excluded from the identified AOI or AOIs. Thus, these areas may not be imaged during the imaging scan, which is undesirable. Conversely, if the imaging scan is performed uniformly over the entire scanning area 20, the total scanning time may be very long and a large amount of data may be generated that is time-consuming to process. Thus, a compromise is found where the entire scanning area 20 is imaged during the imaging scan, but the target scanning speed is faster in the sample-free area 26 than in the sample-containing area 24.

[0030] In step 102 of the method, a seed location is selected within the sample containing area 24 in the xy plane (typically at the edge of the sample containing area 24 within the AOI) and the focal height of the sample is measured at this location using the line scanner 3. The focal height of the adjustable focusing system 4 is varied over a predetermined range of focal heights to acquire image information from the target 6 at several different focal positions with focal depths of up to around 1 μm along the z axis. A focus position is then calculated from the image information at each focus position by using a focus parameter in the form of a focus merit value. Typically, the focus merit value is a numerical measure of the complexity in an image, the higher the value, the more detail there is in the image. A more focused image has a corresponding higher merit value. In this case, the focus height with the highest merit value is stored as the measured focal position for the seed location and the process is repeated for any remaining seed locations identified.

[0031] An image scan path is calculated along which the scan head 2 will be moved end to end such that the line scanner 3 images the entire scan area 20 (comprising the sample-containing area 24 and the sample-absent area 26). The line scanner 3 is then moved to a start location within the sample-absent area 26 and the focus height is set according to the focus height recorded for the focus position relative to the seed location. For example, the focus height at the start location can be the same as the focus height at the seed location or it may be based on an extrapolation of this location.

[0032] The intention of acquiring images of the sample-absent region 26 during the imaging scan is to be able to image a portion of the sample if this portion is located in the sample-absent region 26 (and therefore was not detected based on analysis of the pre-scan images). However, it is desirable that any images of the sample are acquired with a resolution similar or equal to that of the images acquired within the sample-containing region 24. Thus, according to the first embodiment, one or more image parameters are monitored by the computer system 10 for each image acquired along the image scanning path. Typically, the monitored image parameters will change above and below a threshold each time a sample enters or leaves the field of view of the line scanner 3. For example, a focus merit value or frequency content may be monitored at each location, and typically these values ​​will increase above a pre-determined threshold each time a sample enters the field of view. Alternatively or additionally, the brightness of the image may be monitored, where a drop in brightness below a brightness threshold (e.g., 90-95% of maximum brightness) indicates that a sample is entering the field of view of the line scanner 3. A transparent slide will have a given brightness value with fixed noise or minimal high frequency response. If the brightness drops below a brightness threshold or if the noise or high frequency values ​​increase above a corresponding threshold, tissue may be present and the scan speed may be reduced to increase the scan resolution. Typically, this reduction will be over a region to eliminate sudden accelerations. In this way, tissue not detected in the pre-scan image may be scanned with high resolution. When tissue is no longer detected, the movement speed may be increased to reduce the scan time.

[0033] The imaging scan begins at step 103, at which point the scan head 2 is moved from a starting location over an image scan path to acquire images of multiple adjacent locations on the target 6. The image scan path is calculated such that the line scanner 3 moves at a first target speed in the sample-free region 26 and at a second target speed in the sample-containing region 24, with the first target speed being faster than the second target speed. The spatial resolution of the pass-through scan can be changed by increasing the scan speed (also called the "movement speed"). If the scan resolution in the sample-containing region 24 is set to 4000 lpmm x 4000 lpmm, by increasing the movement speed, the scan resolution is changed to 4000 lpmm x ylpmm, where y can be between 4000 lpmm and 1000 lpmm or even lower. Typically, the sample-free region 26 contains only transparent slides, so it is not important to scan this region with high resolution. Thus, if the scanning resolution is selected to be 1000 lpmm, the sample-absent region 26 can be scanned four times faster than the sample-containing region 24. The starting position for the imaging scan is within the sample-absent region 26, and therefore the line scanner 3 is initially moved at a first target speed. However, when a change in one or more image parameters is monitored within the sample-absent region 26 indicating that a sample comes into the field of view of the line scanner 3, the scanning speed is reduced to a second target speed. Thus, on-the-fly detection of the sample can be performed, and the sample is imaged at high resolution as long as it is present on the target 6.

[0034] When the pre-scan data indicates that the imaging scan is approaching the edge of the sample, the scan speed can be reduced so that the edge of the sample is captured with high resolution. This is shown in FIG. 6, where the top graph shows two areas of the target 6, with the clear area on the left representing the sample-free region 26, whereas the shaded area as a marker represents the sample-containing region 24. The bottom graph of FIG. 6 illustrates the change in scan speed as the line scanner 3 moves from imaging the sample-free region 26 to imaging the sample-containing region 24. The image scan path is calculated such that the scan speed gradually decreases from a first target speed to a second target speed before the line scanner 3 moves from imaging the sample-free region 26 to imaging the sample-containing region 24. In this example, the second target speed is half of the first target speed. A first image of the target-containing region 24 is then acquired at the second target speed without any sudden deceleration occurring inside this region. Similarly, as shown by FIG. 7, once the pre-scan data indicates that the edge of the sample-containing region 24 has been passed, the scan speed is increased back to the first target speed. This speed increase is done over the region to reduce the rate of acceleration, creating a smooth transition between the different regions. There will be a maximum acceleration / deceleration and a maximum rate of acceleration change. Taking the example where the target 6 is moved and the line scanner 3 is held stationary during the imaging scan, it would require only 3 J to change the speed of a 5 kg stage and slide from that required for a 1000 lpmm scan to that required for a 4000 lpmm scan, but it would require 85 W to make this change within the time of a single line. Spreading it over 10 lines would require the same energy, but the power would be reduced to 8.5 W.

[0035] The vertical dashed lines in the upper graphs of Figures 6 and 7 illustrate where image capture is triggered. The line scanner 3 acquires images at a constant rate throughout the imaging scan. Thus, the spatial density of sampling points (i.e., where image capture is triggered) increases when the scanning speed decreases from a first target speed to a second target speed (Figure 6), and then decreases when the scanning speed increases from the second target speed to the first target speed (Figure 7). In the absence of any interpolation, this scanning speed change will lead to a corresponding image resolution change between the sample-free region 26 and the sample-containing region 24, as will be discussed below with reference to Figure 9.

[0036] In a first embodiment, the scanning speed is held constant at the second target speed throughout the sample-containing region 24. However, in other embodiments, the scanning speed can be increased if one or more monitored image parameters indicate that a sample is not located within the sample-containing region 24. It is believed that this may occur, for example, if the AOI detection algorithm erroneously identifies a surface marking on the target 6 as part of the sample. The depth of focus of the line scanner 3 is typically less than 3 micrometers, i.e., the surface marking is unlikely to be identified as a sample in the imaging scan. Optionally, the scanning speed can be increased from the second target speed only if a sample is not detected within the sample-containing region 24 within a threshold distance (or equivalently after a threshold number of lines have been acquired). The scanning speed can then be increased from the second scanning speed to the first scanning speed or optionally to a value between these two values. For example, the increase in scanning speed can be balanced by the probability that a sample is present at a given location or region as determined by a probability map. That is, if the preliminary scan analysis provides good certainty of the presence of a sample, the scan speed would not be increased because it is expected that a sample will be imaged in sample-containing region 24 and it is desirable to image the sample with the highest resolution when it is reached. However, if the probability map indicates only a relatively low certainty that a sample is present at a given location within the area marked as a sample-containing region (i.e., the preliminary scan analysis was uncertain as to the presence of a sample in that region), the scan speed could be increased by a factor. For example, if there is only a 50% certainty that a sample is present at a given location, the scan speed would be increased by only 50% of its possible increase.

[0037] In yet another embodiment, the scanning speed is continuously adjusted during the imaging scan as a percentage of the certainty of finding a sample at a given location determined from a probability map generated from the pre-scan data. Alternatively, it is contemplated that the speed can be set for brightness values ​​with a linear ramp within this range, where a threshold brightness of 95% indicates a transparent slide (resolution set to 1000 lpmm) and a second threshold brightness of 90% indicates a sample (resolution set to 4000 lpmm). For example, if the brightness is measured to be 92.5%, the scanning speed can be set to generate a resolution of 2500 lpmm.

[0038] Returning to the first embodiment, when the line scanner 3 proceeds from acquiring images of the sample-containing area 24 to acquiring images of the sample-free area 26 again, the scanning speed is increased to a first target speed (illustrated by FIG. 7). In case of a change in one or more monitored image parameters indicating the presence of a sample in the field of view of the line scanner 3, the scanning speed is reduced again to a second target speed so that the sample is imaged with a suitably high resolution. In this way, if a part of the sample is not detected by the analysis of the pre-scan data, a high-resolution image of this part will still be acquired. FIG. 8 illustrates such an example where a part of the sample is detected in the sample-free area 26. The upper graph illustrates the scanning speed as the line scanner 3 moves along the image scanning path. From each image acquired along the image scanning path, an image parameter in the form of a focus merit value is calculated. The focus merit value for each image or "line" is marked with a "x" at the corresponding position along the image scanning path in the lower graph of FIG. 8. The scanning speed is held at the first target speed for successive image captures where the focus merit value is lower than the focus merit value threshold. At the location marked "A", the focus merit value is not higher than the threshold, indicating that a portion of the sample is visible in the image for that location (inside the sample-absent region 26). In response to this change in focus merit value, the scanning speed is reduced from the first target speed to a second target speed. The line scanner 3 is decelerated over the following three lines until the second target speed is reached. The focus merit value is approximately constant throughout the portion of the image scanning path that extends from position A to position B, illustrating the extension of the sample over this region. The detection of a sample in the so-called sample-absent region illustrates how analysis of only pre-scan data can provide inaccurate results and therefore why it is advantageous to acquire an image of the sample-absent region 26 in addition to the sample-containing region 26.In the first embodiment, the scan rate is adjusted on the fly in response to changes in the monitored image parameters, while in an alternative embodiment, the scan rate can be set based on analysis of pre-scan data only, with sample-containing regions being imaged at a slower rate than sample-absent regions.

[0039] Throughout the imaging scan, an estimated focus level can be calculated using techniques known in the art, such as those described in US 9116035 B2. When the sample is within the field of view of the line scanner 3, the focal height of the line scanner 3 is adjusted between each xy location along the image scan path towards or to the estimated focus position. The target 6 is imaged in this manner within one or more scan widths, following a serpentine or raster image scan path, until the scan area is completely imaged.

[0040] In step 104, the individual images acquired by the line scanner 3 are combined with each other to form a high-resolution image of the target. It is desirable that the observation resolution of the output image is uniform for both the fast and slow scan areas. The image resolution in the areas scanned at the higher scan speed can be increased by using an interpolation technique such as bicubic interpolation or other image processing techniques known in the art (e.g., as described in Chapter 3 of "Numerical Recipes in C: the Art of Scientific Computing", Cambridge University Press, 1992). That is, it is possible to scan at a lower resolution but generate what appears to be a high-resolution image. In step 104, the interpolation ratio will be appropriately adjusted according to the scan speed at which the images were acquired in order to maintain a constant resolution in the output high-resolution image. This will be further discussed below with reference to FIG. 9.

[0041] FIG. 9 is the process of FIG. 6, but in this case the upper graph illustrates the amount of light incident on the line scanner 3. The light level is initially high (approximately 0.9 on the normalized scale shown) and is approximately constant over the area of ​​the target 6 where no sample is present. The light level then drops to below 0.1 (inside the sample-containing region 24) when the sample enters the field of view of the line scanner 3. The light level is monitored from the images captured along the image scanning path, and the locations of the sampling points are marked with circles. The spatial separation between adjacent sampling points is approximately twice as wide in the area marked "C" (in the sample-free region 26) as in the area marked "E" (in the sample-containing region 24). The area marked "D" illustrates the area where the scanning speed falls between the first and second target speeds. In performing step 104, the image data from the line scanner 3 is interpolated onto the nominal HR grid to generate a constant resolution over the image scanning path. The interpolated data is marked with stars in the upper graph. As can be seen, image data has been interpolated between adjacent sampling points for regions C and D. The high resolution image generated by the imaging scan can then be displayed to a user by computer system 10 for sample analysis.

[0042] An improved method for operating a microscope scanner is thus provided which ensures that the entire sample is imaged with high resolution and without unduly extending the overall scan time. [Explanation of symbols]

[0043] 100 Performing a preliminary scan 101 AOI Identification Stage 102 measuring the focal height at the seed location 103 Acquiring an image along an image scanning path 104 generating a high resolution image of the target

Claims

1. 1. A method of operating a microscope scanner, comprising: generating pre-scan data on a target (6) comprising a sample, obtaining a pre-scan image of the target from a pre-scan (100); and identifying (101) one or more sample-containing regions (24) of the target (6) corresponding to locations on the target where the sample is expected to be present and one or more sample-absent regions (26) corresponding to locations on the target where the sample is expected to be absent from the target; the generating step comprising: performing an imaging scan of a scan area (20) of the target (6) including the one or more sample-containing areas and the one or more sample-absent areas (26); Equipped with The microscope scanner (1) comprises a line scanner (3), The step of performing an imaging scan includes: moving (103) the line scanner (3) relative to the target (6) along an image scanning path and acquiring (103) an image of the target at each of a plurality of locations along the image scanning path using the line scanner (3); adjusting a scan speed of the line scanner (3) along the image scan path based on the pre-scan data such that the target (6) is imaged at a higher scan speed in the one or more sample-free regions (26) than in the one or more sample-containing regions (24); monitoring one or more image parameters from the images of the target acquired along the image scan path; further adjusting the scanning speed of the line scanner (3) within the one or more sample absent regions (26) in response to changes in the one or more image parameters; Equipped with the one or more image parameters comprise a focus merit value, where a more focused image has a higher focus merit value; wherein the scan speed of the line scanner is reduced in response to the focus merit value increasing.

2. adjusting the scan speed based on the pre-scan data comprises imaging the target (6) in the one or more sample-free regions (26) at a first target speed and imaging the target in the one or more sample-containing regions (24) at a second target speed, the first target speed being greater than the second target speed; 2. The method of claim 1, wherein the imaging scan further comprises reducing the scanning speed from the first target speed to or towards the second target speed before the line scanner transitions from acquiring an image of the sample-free region to acquiring an image of the sample-containing region.

3. 3. The method according to claim 1 or 2, characterized in that the imaging scan further comprises the steps of calculating the focus merit value at each said location along the image scan path, and adjusting the focus height of the line scanner (3) along the image scan path based on the focus merit values.

4. the images are acquired at a constant rate in time during the imaging scan; The method according to any one of claims 1 to 3, characterized in that the line scanner (3) is moved continuously along the image scanning path relative to the target (6) during the imaging scan.

5. the depth of focus for said pre-scan is greater than 500 micrometers; The method of any one of claims 1 to 4, wherein the depth of focus for each image acquired along the image scan path is less than 3 micrometers.

6. The method of any one of claims 1 to 5, wherein the pre-scan image is acquired from a single pass of a pre-scan camera (12) along a pre-scan imaging path.

7. generating pre-scan data further comprises performing image analysis of the pre-scan images to generate a probability map indicative of the likelihood that the sample is present at each location on the target; 7. The method of any one of claims 1 to 6, wherein the one or more sample-containing regions (24) and the one or more sample-absent regions (26) are selected from the probability map.

8. 8. The method of claim 7, wherein adjusting the scanning speed of the line scanner (3) along the image scanning path based on the pre-scan data comprises adjusting the scanning speed according to the likelihood that the sample is present at a given location on the target as determined by the probability map.

9. the one or more sample-absent regions (26) correspond to locations from the probability map that are unlikely to contain the sample; the one or more sample-containing regions (24) correspond to locations from the probability map that are likely to contain the sample; 9. The method of claim 7 or 8, wherein generating pre-scan data further comprises identifying one or more areas of uncertainty on the target from the probability map having a probability of containing the sample that is between the probability of the sample-free areas and the sample-containing areas containing the sample.

10. adjusting the scan rate based on the pre-scan data comprises imaging the target (6) at a first target rate in the one or more sample-absent regions (26), imaging the target at a second target rate in the one or more sample-containing regions (24), and imaging the target at a third target rate in the one or more regions of uncertainty; 10. The method of claim 9, wherein the third target velocity is greater than the second target velocity and less than the first target velocity.

11. generating (104) a high resolution image of the target (6) from a combination of the images acquired during the imaging scan; 11. A method according to any one of claims 1 to 10, wherein generating the high resolution image comprises increasing the resolution of an image acquired at a higher scanning rate using image interpolation to correspond to the resolution of the image acquired at a lower scanning rate.

12. 12. The method of claim 11, wherein generating (104) the high resolution image comprises adjusting an interpolation ratio to maintain a constant resolution in the high resolution image.

13. The imaging scan is performed in transmission mode for the microscope scanner (1), The method according to any one of claims 1 to 12, characterized in that the microscope scanner is a bright field microscope.

14. A method according to any one of claims 1 to 13, further comprising a step of selecting the scanning area (20) from the pre-scan image so as to cover substantially the entire target (6) excluding any opaque areas (22) of the target.

15. A computer program product comprising instructions which, when executed with a microscope scanner (1) comprising a line scanner (3) and a target (6) comprising a sample, causes the microscope scanner to perform the method according to any one of claims 1 to 14.

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