Patent Publication Number: US-2007124085-A1

Title: Method of processing a biological image

Description:
FIELD OF THE INVENTION  
      The invention relates to a method of processing an image of a biological sample containing nuclei, cytoplasm and micronuclei. The invention further relates to a computer programme performing the method, a data carrier comprising the computer programme and a system arranged to run the computer programme.  
     BACKGROUND OF THE INVENTION  
      The processing of images is of particular relevance in the field of biology, where images of biological samples are to be analysed for the presence of certain features. The features may include intracellular components, fibres, and granules. When using fluorescent microscopy, the distribution of elements in the samples labelled with a fluorophore can be imaged and stored as intensity values of pixels in a digital image. Different elements can be labelled with different fluorophores, which allows imaging a specific element by choosing an appropriate wavelength for illumination of the sample and an appropriate filter for collecting the radiation from the sample. A specific combination of wavelength and filter is called a channel. For example, a first channel may be set for imaging nuclei of cells and a second channel for imaging cytoplasm surrounding the nuclei.  
      In the field of drug discovery the images of a large number of biological samples have to be processed. The method of processing must provide for a fast execution on a computer to allow handling of a complete assay in a reasonable time. A particular drug discovery assay looks at micronucleus induction for finding genotoxic compounds. Analysis of micronucleus formation is an important component of toxicology evaluation of new drug candidates and other chemicals and materials, such as food dyes and cosmetics that are intended for human consumption or use or which may be indirectly or accidentally consumed or ingested. The analysis determines the incidence of micronuclei in the cytoplasm of cells and, preferably, the attribution of the micronuclei with mononucleate cells and binucleate cells.  
      Known automated methods of analysing the incidence of micronuclei are relatively slow. One of the known methods uses an image from a first channel to locate nuclei in the sample, identifies the cytoplasm in an image from a second channel and segments the cytoplasm from the background by a watershed method, and, subsequently, determines the presence of micronuclei in the area of the cytoplasm in the image from the first channel. The segmentation of the cytoplasm and the use of the second channel require a substantial amount of time. It is an object of the invention to provide a faster method.  
     SUMMARY OF THE INVENTION  
      The object of the invention is achieved in a method of processing an image of a biological sample containing nuclei, cytoplasm and micronuclei, each of the nuclei being surrounded by cytoplasm, the cytoplasm having a maximum distance, which is the largest extent of the cytoplasm from its nucleus occurring in the image, the method including the steps of setting a search distance at a value less than the maximum distance, searching for micronuclei within the search distance from the nuclei and annotating the micronuclei found in the search.  
      The method according to invention avoids the use of the watershed segmentation and can operate on the image of a single channel. The maximum distance is the largest value of the extent of the cytoplasm from the nucleus which it surrounds occurring in the image. A search is carried out for micronuclei having a distance of less than the second distance from the edge of a nucleus. Such micronuclei are in general located in the cytoplasm surrounding the nucleus. Micronuclei complying with this requirement are annotated as a find of the search. The method allows the attribution of micronuclei to specific cells, such as mononucleate cells and binucleate cells.  
      Although micronuclei are in general located relatively close to the edge of the nucleus, micronuclei that are located in the cytoplasm at a distance larger than the second distance may be discarded by the method according to the invention. This does not affect an analysis based on relative incidences, for example an analysis focused on the increase of micronuclei incidents as a function of the concentration of a genotoxic compound. When absolute instances are acquired, reference samples with known incidences may be used to scale the incidences obtained by the method according to the invention.  
      In a first preferred embodiment of the method it includes the step of defining a first area around at least one of the nuclei, the first area being enclosed between an inner boundary and an outer boundary, the distance between an edge of the first element and the outer boundary being substantially equal to the search distance, and the search for micronuclei being carried out within a search area restricted by the first area. The shape of the search area is such that it follows the edge of the nucleus. The search for micronuclei will in general extend to all nuclei in the entire image. The computation of the search areas can be done substantially faster than the prior art determination of the extent of the cytoplasm with the watershed method. The search for micronuclei in the search areas around the nuclei in the image can be carried out in the same channel as the identification of the nuclei and can also be performed relatively fast.  
      In an alternative method the search distance is set at a value substantially equal to half the diameter of the nucleus to which the search area pertains. The word ‘substantially’ means that the value is preferably between 0.3 and 0.7 times the diameter. When the shape of the nucleus is not circular, the diameter can be calculated as the average of the longest and shortest diameter of the nucleus. The diameter may also be averaged over the nuclei to be investigated.  
      In a special embodiment the inner boundary of the search area is the edge of the nucleus. Since the edge of the nucleus has already been determined in the identification of the nuclei, the determination of the inner boundary does not require more calculations. In another preferred method the inner boundary of the first area is arranged outside the nucleus and inside the outer boundary. A dilation of the inner boundary from the edge of the nucleus avoids that variations in staining intensity at the edge are incorrectly identified as micronuclei. When the outer boundary is defined in terms of a certain distance from the inner boundary, the search distance is equal to this distance plus the distance between the inner boundary and the edge of the nucleus.  
      The method may be refined by the step of defining a second area around the nucleus, the second area being a region of overlap of the first area and the area of the cytoplasm surrounding the nucleus, the search area being restricted by the second area. This prevents the search area from falling outside of the region of the cell and avoids annotating objects outside cells as micronuclei. The extent of the cytoplasm can be determined in a second channel, different from the channel used for identifying the nuclei and micronuclei, where a fast-executing thresholding may be used to identify the cytoplasm.  
      When nuclei are relatively close, a search area surrounding one nucleus may overlap with part of a neighbouring nucleus. To avoid identification of the area of overlap as a micronucleus, the search area is preferably restricted to the first or second area excluding the region of overlap with neighbouring nuclei.  
      It should be noted that the U.S. Pat. No. 5,989,835 discloses a method of locating reporter molecules in cells. The method defines a nuclear mask within the area of a cell nucleus and a sampling area around the nucleus, both in an image of a first channel. The presence of reporter molecules is determined in an image of another channel by measuring the average fluorescence in the nuclear mask and the sampling area. The patent does not disclose the use of a search area for determining the presence of discrete objects such as micronuclei. It neither discloses a search for objects in an image from the same channel as the one in which the areas are defined, providing a considerable advantage in processing speed, nor a search area close to the nucleus, where it is most likely to find micronuclei.  
      In a second preferred embodiment of the method it includes the step of determining a third distance between at least one of the micronuclei and the nearest nucleus, and annotating the micronucleus when the third distance is less than the search distance. The method searches the image for objects having the size and intensity of a micronucleus, determines the distance to the nearest nucleus and annotates the micronucleus as a find only when the distance is less than the search distance. The method of this second preferred embodiment can execute very fast and is simple to implement.  
      In an alternative method the search distance is set at a value substantially equal to half the diameter of the nuclei. The word ‘substantially’ means that the value is preferably between 0.3 and 0.7 times the diameter. When the shape of the nucleus is not circular, the diameter can be calculated as the average of the longest and shortest diameter of the nucleus. The diameter is preferably averaged over the nuclei to be investigated.  
      To prevent a micronucleus from being located outside a cell, the method preferably includes the step of annotating the micronucleus only when it overlaps the cytoplasm surrounding the nearest nucleus. The extent of the cytoplasm can be determined as set out above.  
      A second aspect of the invention relates to a computer program arranged to perform the method of the invention.  
      A third aspect of the invention relates to a data carrier in which the computer program is stored.  
      A fourth aspect of the invention relates to a system, preferably an analyzer system, including a processing unit for running the computer program. The system includes preferably a data carrier in which the computer program is stored.  
      Further features and advantages of the invention will become apparent from the following description of preferred embodiments of the invention, given by way of example only, which is made with reference to the accompanying drawings. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       FIG. 1  shows the formation of a micronucleus during cell division.  
       FIG. 2  shows a schematic view of a fluorescence microscope used to image samples.  
       FIG. 3  shows a schematic illustration of data processing components of a system.  
       FIG. 4  shows a flow diagram of a first preferred embodiment of the method of processing an image according to the invention.  
       FIG. 5  shows schematically nuclei with search areas.  
       FIG. 6  shows a flow diagram of a second preferred embodiment of the method of processing an image according to the invention.  
       FIG. 7  shows schematically nuclear objects. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION  
      Micronucleus induction is a key characteristic of genotoxic compounds. The analysis of micronucleus formation is therefore an important object of investigation. Micronuclei formation occurs during cell division of cells exposed to genotoxic compounds. This may be the result of either DNA strand breakage due to clastogenic compounds or of erroneous chromosome segregation caused by interference with components of the cell&#39;s chromosome separation machinery, such as tubulin, due to aneugenic compounds.  
       FIG. 1  shows the formation of a micronucleus during cell division, also called mitosis. A cell  1  comprises a nucleus  2  surrounded by cytoplasm  3 . The cell during division is represented by reference numeral  4 . The divided chromosomes  5  are separated by microtubules  6 . The Figure shows a DNA strand breakage  7  and a breakage  8  of a microtubule. These defects in the chromosome segregation result in chromosome fragments or whole chromosomes. After cytokinesis, i.e. the splitting of the cell into two daughter cells each comprising a set of segregated chromosomes, there are two cells  9 ,  10  each having a nucleus  11 ,  12 . The chromosome fragments or whole chromosomes caused by the defect reside as a micronucleus  13  in the cytoplasm of cell  10 . A cell containing a single nucleus, such as cells  9  and  10  in  FIG. 1 , are called mono-nucleate cells. A cell in which no cytokinesis has taken place after mitosis and containing two nuclei within the cytoplasm is called a bi-nucleate cell.  
      In-vitro micronucleus assays are performed by incubating cells with test compounds for a sufficient period to allow one or more rounds of cell division to occur to allow the formation of micronuclei where test compounds have clastogenic or aneugenic activity. Since cell division is a requirement for micronucleus formation, it is necessary to ensure that cells are actively dividing during the course of the assay. A number of approaches are available to achieve confirmation of cell division, including the use of cytokinesis blockage using cytochalasin B. In protocols employing cytochalasin B inhibition of cytokinesis, addition of the inhibitor arrests cells immediately prior to separation of daughter cells, resulting in the formation of bi-nucleate cells. Consequently analysis of a cell population treated with a test compound for the relative frequency of mononucleate and binucleate cells provides a measure of the proliferative activity of the culture. This proliferation index can be used to identify compounds which cause cell cycle delay or arrest, and to indicate the need to retest compounds where there is a risk of a false negative assay result arising from compound cytotoxicity. For guidance on proliferation controls and use of cytokinesis blockage, see the ‘Report from the in-vitro micronucleus assay working group’ by M. Kirsch-Volders et al in the journal Mutat. Res. 2003, Vol. 540(2), page 153-163.  
      The presence of micronuclei can be readily detected using standard DNA staining methods and fluorescence imaging. Common stains for DNA are Hoechst™ 33342 and DRAQ5™. Cytoplasm can be stained using FITC or Calcein blue AM. Non-viable cells can be detected by using the stain Propidium iodide. The samples for the assay are prepared in a conventional way. Samples containing different amounts of the test compound may be deposited in the wells of a microtiter plate.  
       FIG. 2  shows a schematic view of a fluorescence microscope which can be used to image the above samples in an analyzer system such as the GE Healthcare IN Cell Analyzer 3000 system, disclosed in U.S. Pat. No. 6,400,487 and U.S. Pat. No. 6,388,788. The microscope comprises a source of electromagnetic radiation, for example a light bulb  21  and/or a laser  22  emitting radiation in the optical range, 350-750 nm, which is collimated by lenses  23  and  24 , respectively. The microscope further comprises a cylindrical lens  25 , a first slit mask  26 , a first relay lens  27 , a beam splitter  28  in the form of a dichroic mirror, an objective lens  29 , a microtiter plate  30  containing a two-dimensional array of sample wells  31 , a tube lens  32 , a filter  33 , a second slit mask  34  and a detector  35 . These elements are arranged along the optical axis OA defined by slit apertures  36 ,  37  in masks  26 ,  34 , respectively, and extending perpendicular to the plane of  FIG. 2 . The focal lengths of lenses  27 ,  29  and  32  and the spacings between these lenses as well as the spacings between mask  26  and lens  27 , between objective lens  29  and microtiter plate  30  and between lens  32  and mask  34  are such as to provide a confocal microscope.  
      In this embodiment, the electromagnetic radiation from the source is focused to a line using the cylindrical lens  25 . The shape of the line is optimized by the first slit mask  26 . The slit mask  26  is shown in a plane of the optical system that is conjugate to the plane of the microtiter plate  30 . The illumination stripe formed by the aperture  36  in the slit mask  26  is relayed by lens  27 , dichroic mirror  28  and objective lens  29  onto the microtiter plate  30 . For convenience of illustration, the optical elements are depicted in cross-section and the well plate in perspective. The projection of the line of illumination onto well plate  30  is depicted by a line  38 . As indicated by arrows A and B, well plate  30  may be moved in two directions (x, y) parallel to the directions of the array by means not shown.  
      Alternatively, the slit mask  26  may be arranged in a Fourier plane of the optical system, which is in a plane conjugate to the back focal plane (BFP)  39  of the objective lens  29 . In this case the slit aperture  36  lies in the plane of the figure, the lens  27  relays the illumination stripe formed by the aperture  26  onto the back focal plane  39  of the objective  29 , which transforms it into a line in the plane of the microtiter  30  perpendicular to the plane of  FIG. 1 .  
      The radiation from the source may also be focused into the back focal plane  39  of the objective lens  29  without use of the slit mask  26 . This can be accomplished by the combination of the cylindrical lens  25  and the spherical lens  27  as shown in  FIG. 2 , or the illumination can be focused directly into the plane  39  by the cylindrical lens  25 .  
      An image of the sample area, for example a sample present in the sample well  31 , is obtained by positioning the microtiter  20  such that the line  38  of illumination is arranged across the sample, imaging the fluorescence emission from the sample onto detector  35  and translating the plate  30  in a direction perpendicular to the line of illumination, synchronously with the reading of the detector  35 . The fluorescence emission is collected by the objective lens  29 , projected through the beam splitter  28 , and imaged by lens  32  through filter  33  and the second slit mask  34  onto the detector  35 , such as is appropriate to a confocal imaging system having an infinity-corrected objective lens  29 . The beam splitter  28  and filter  33  preferentially block light at the illumination wavelength. The detector  35  may be a CCD array; the detector may be either one dimensional or two dimensional. If a one dimensional detector is used, slit mask  34  is not required. The illumination, detection and translation procedures are continued until the prescribed area has been imaged. Mechanical motion of the microtiter is simplified if it is translated at a continuous rate. Continuous motion is most useful if the camera read-time is small compared to the exposure-time. In a preferred embodiment, the camera is read continuously. The displacement d of the sample during the combined exposure-time and read-time may be greater than or less than the width of the illumination line W, exemplarily 0.5 W ≦d ≦5 W. All of the wells of a multi-well plate can be imaged in a similar manner. However, it will be recognized that a non-confocal microscope can also be used, e.g. as incorporated in the GE Healthcare IN Cell Analyzer 1000 system, disclosed in U.S. Pat. Nos. 6,563,653 and 6,345,115. Other embodiments of fluorescence microscopes that may be used to acquire the images are disclosed in the international patent application publication number WO00/235474.  
       FIG. 3  shows a schematic illustration of data processing components of an analyzer system. The system includes a cell analysis system  40 , based on the GE Healthcare IN Cell Analyzer system. The cell analysis system  40  includes detector D 1 , which may be a detector  35  of a microscope as shown in  FIG. 2 . The cell analysis system  40  further comprises a control unit  41 , an image data store  42  and an Input/Output (I/O) device  33 .  
      An associated computer terminal  44  includes a central processing unit (CPU)  45 , memory  46 , a data storage device such as a hard disc drive  47  and I/O devices  48  which facilitate interconnection of the computer with the cell analysis system  40  and interconnection of the computer with a display element  49  of a screen  50  via a screen I/O device  51 , respectively. Operating system programs  60 , such as Microsoft Windows 2000™ or Windows XP™, are stored on the hard disc drive  47 , and control, in a known manner, low level operation of the computer terminal  44 . Program files and data  61  are also stored on the hard disc drive  47 , and control, in a known manner, outputs to an operator via associated devices and output data stored on the hard disc drive. The associated devices include the display  49  as an element of the screen  50 , a pointing device (not shown) and a keyboard (not shown), which receive input from, and output information to, the operator via further I/O devices (not shown). Included in the program files  61  stored on the hard disc drive  47  are an image processing and analysis application  62 , an assay control application  63 , and a database  64  for storing image data received from the cell analysis system  40  and output files produced during data processing. The image processing and analysis application  52  includes image processing and analysis software packages. A method according to an embodiment of the invention may be implemented as software within the image processing and analysis application  62 .  
      The performance of a scan using the cell analysis system  40  is controlled using control application  63 , and the image data are acquired. In an embodiment, the control application acts in concert with an autofocus system of the microscope shown in  FIG. 2 . After the end of acquisition of image data for at least one well in a microtiter plate by the detector D 1 , the image data are transmitted to the computer  44  and stored in the database  64  on the computer terminal hard disc drive  47 , at which point the image data can be processed using the image processing and analysis application  62 .  
       FIG. 4  shows a flow diagram of a method of processing an input image of a biological specimen according to the first preferred embodiment. The image being processed first is taken from the channel pertaining to the fluorophore used to label the nuclei. In the first step  70  of the method the nuclei are segmented. The segmentation may be carried out using thresholding, which is a relatively fast procedure. The intensity of an object in the image is an indicator of the DNA content of the object. The threshold for the intensity is set by the operator of the analyzer system at a value that clearly reveals the nuclei. An object is labelled as a nucleus when it has an intensity above the threshold intensity and a size larger than an operator-set minimum threshold size and less than a maximum threshold size. The minimum threshold size is usually set at ⅓ of the average size of the nuclei, thereby avoiding that micronuclei are identified as nuclei.  FIG. 5  shows schematically an image of a biological sample in which two objects  80  and  81  are identified as nuclei.  
      In the second step  71  of the processing the cells found in the image are classified. Micronucleus assays usually distinguish mononucleate and binucleate cells. Mononucleate objects have a higher form factor than binucleate objects. A threshold form factor is set by the operator of the analyzer system. The operator may use a plot of the form factor versus the intensity to set the threshold at the optimum value. The form factor is the ratio of the shortest diameter of an object over its longest diameter Nuclei having a form factor larger than the threshold are labelled as mononucleate and nuclei having a lower form factor are labelled as binucleate. Nuclei that do not comply with specific requirements, such as the requirement that the cell must be alive, may not be labelled as mono- or binucleate objects and are excluded from further processing. Nucleus  80  in  FIG. 5  is classified as mononucleate and nucleus  81  as binucleate.  
      The third step  72  of the processing is an optional step in which the extent of the cytoplasm surrounding the nucleus is established. The extent of the cytoplasm is determined in an image of a second channel pertaining to the marker of the cytoplasm. The area of the cytoplasm may be determined by thresholding the image using an operator-set threshold. The threshold is set by visual inspection making the assigned edge of the cytoplasm a reasonable approximation of the visible cytoplasm.  
      The search area  82 ,  83  around each labelled nucleus  80 ,  81  is defined in the fourth step  73 . The search area is equal to a first area having an inner boundary and an outer boundary. The inner boundary may be the edge  84 ,  85  of the nucleus. The inner boundary may also extend beyond the edge of the nucleus into the surrounding cytoplasm by dilating the edge by a few pixels. The dilation is small compared to the size of the nucleus, typically 1-3 pixels for a nucleus having a maximum diameter between 10 and 30 pixels. The size of the nucleus measured in pixels depends on the cell type and the analyzer system; for CHO cells on the IN Cell 3000 system a nucleus is around 10 to 15 pixels maximum diameter and a double nucleus such as found in bi-nucleate cells 20 to 30 pixels. If erosion is previously used to define the area of the nucleus, the dilation must be by a higher value to ensure that the search area is in the cytoplasm. The extended inner boundary is shown in  FIG. 5  as elements  86  and  87 .  
      The outer boundary must be determined such that the distance between the outer boundary and the edge of the nucleus is equal to an operator-set search distance ds. The search distance is restricted by the maximum distance of the cytoplasm in the image. The nuclei  80  and  81  in  FIG. 5  are surrounded by cytoplasm  88  and  89 , respectively. The largest extent of cytoplasm  89  is indicated by the line d 1 . The longest length of line d 1  of the labelled nuclei present in the area of interest of the image is called the maximum distance. The search distance must be smaller than the value of the maximum distance. The search distance must neither be too small, which will cause missing too many micronuclei in the cytoplasm, nor too large, thereby annotating objects outside the cytoplasm. A search distance equal to half the average diameter of the nucleus is a typical setting for many samples, i.e. a diameter of the outer boundary equal to twice the diameter of the nucleus. The outer boundary is set in practice by dilating the inner boundary or the edge of the nucleus. In many practical cases the search distance is equal to 11 pixels. This setting can also be used as a first setting, from where the search distance may be optimised. The value of the search distance may be determined in various ways, for example it may be taken from a database or be determined using an overlay of the search area with the cytoplasm.  
      The search area may, optionally, be restricted by a second area which overlays the cytoplasm. The bitmap of the cytoplasm may be overlaid with the image containing the search areas. Any area  90 ,  91  of the search area falling outside the area of the cytoplasm is removed from the search area. The bitmap may also be used to visualize the relation between the outer boundary of the search area and the extent of the cytoplasm. Another way of restricting the search area is to overlay the intensity image of the cytoplasm with the image containing the search areas. Any area of the first area  82 ,  83  that has a cytoplasm intensity lower than an operator-set minimum marking threshold is excluded from the search area, resulting in a second area. The search area is restricted by the second area.  
      The search area may be further restricted in situations where the density of cells is so large that the search area of a nucleus overlaps the area of one or more neighbouring nuclei. Such an area of overlap might falsely be identified as a micronucleus. In an overlay of an image of the nuclei and the search areas, all areas of overlap can be excluded from the search area.  
      Cells in areas of the image having a high density of cells may be excluded from the micronucleus search because of the increased risk of false identification of objects as mono-nucleate cells and bi-nucleate cells.  
      An optional restriction of the cells being processed involves the exclusion of non-viable or dead cells. The viability of cells is determined in an image of a third channel pertaining to the Propidium iodide marker. Any cell having an average intensity between an operator-set minimum value and an operator-set maximum value are identified as dead and cells having an average intensity outside the range are identified viable. The dead cells are excluded from further processing and the search is restricted to living cells.  
      The fifth step  74  of the processing identifies the micronuclei. An object within the search area having an intensity larger than an operator-set threshold intensity and falling between an operator-set minimum size value and an operator-set maximum size value is annotated as a micronucleus.  
      Since each search area belongs to a particular nucleus, the micronuclei found during the search can also be attributed to nuclei. This allows attribution of micronuclei to mono-nucleus cells and to bi-nuclei cells, which can advantageously be used in the toxicity analysis of the test compound.  
      When the search areas overlap, a micronucleus found in the area of overlap can be attributed to the first nucleus in which cytoplasm it is discovered. This attribution depends on the method of scanning the search areas of the nuclei. One method is to scan the image line-wise first from left to right and then from top to bottom.  
      Another method of solving the problem of overlapping search areas is to construct a dividing line between the neighbouring nuclei at equal distance from the edges of both nuclei. The search areas of both nuclei are restricted to the first or second area excluding the area transgressing beyond the dividing line. This definition of the search area also reduces the risk of attribution of a micronucleus to an incorrect nucleus.  
       FIG. 6  shows a flow diagram of a method of processing an input image of a biological specimen according to the second preferred embodiment. The image being processed is taken from the channel pertaining to the fluorophore used to label DNA. In the first step  100  the nuclear objects are segmented. The segmentation may be carried out using thresholding. The threshold for the intensity is set by the operator of the analyzer system at a value that clearly reveals the nuclei and micronuclei. An object is labelled as a nuclear object when it has an intensity above the threshold intensity and a size larger than a set threshold size.  FIG. 7  shows four nuclear objects  80 ,  81 ,  110  and  111 .  
      The second step  101  classifies the objects according to their size and form factor. Objects having a size above an operator-set threshold are classified as nuclei, those having a size below the threshold as potential micronuclei. The threshold may have the value of one third of the average size of a nucleus. The nuclei can be further classified according to their form. The operator may use a plot of the form factor versus the intensity to set the threshold at the optimum value.  FIG. 7  shows two nuclei  80  and  81 , the first one mononucleate and the second one binucleate, and two potential micronuclei  110  and  111 .  
      In a third, optional, step  102  the cytoplasm is segmented in a way as described in the third step  72  of  FIG. 4 .  
      The fourth step  103  determines the distances between the micronuclei and the nuclei. The determination of the distance may be carried out by dilating the edge of the potential micronucleus, until one of the pixels in the dilating edge overlaps an object classified as a nucleus; the distance can be taken as the distance from the overlapping pixel to the edge or the centre of the micronucleus.  FIG. 7  shows the distance d 3  between potential micronucleus  110  and the nearest nucleus  81  and the distance d 4  between potential micronucleus  111  and the nearest nucleus  80 . Alternatively a bounding box, circle or polygon may be applied to the potential micronucleus and enlarged in pixel increments to determine the distance to neighbouring nuclei by expansion of the bounding shape until it overlaps an object classified as a nucleus. In a further variation the dilation of the area around the potential micronucleus, and hence the search for the nearest nucleus of this micronucleus, is stopped when the distance between the edge of the potential micronucleus and the edge of the dilated area is equal to the search distance.  
      If the extent of the cytoplasm has been determined, the search can be refined. An overlay of the cytoplasm and the micronuclei allows the exclusion of potential micronuclei that are located outside the cytoplasm, such as potential micronucleus  111  in  FIG. 7 . In that case only potential micronucleus  110  in  FIG. 7  will be used in the subsequent processing of the image.  
      Similarly, potential micronuclei in areas having a high density of cells may be excluded from the search because of the increased risk of false identification. Dead cells may also be excluded in a way as described for the method of  FIG. 4 .  
      In the fifth step  104  the micronuclei are identified. When the distance between a potential micronucleus and the nearest nucleus is less than the search distance, the potential micronucleus is annotated as micronucleus. When the distance is larger, the potential micronucleus will not be annotated. The search distance is determined as set out in the fourth step  73  of the method shown in  FIG. 4 . The micronucleus can be attributed to the nearest nucleus. Also when the nuclei are relatively close together, the method automatically attributes the micronucleus to the nearest nucleus.  
      The attribution may be refined by finding the nearest nucleus and the next nearest nucleus. The micronucleus is now attributed to the weighted nearest nucleus, where the weighing is according to the integrated intensity of the cytoplasm of the nucleus. The larger the extent of the cytoplasm, the more likely it contains the micronucleus.  
      Although the above methods use thresholding as segmentation method, other methods of segmentation such as the top-hat transform may also be used. Similarly, the second embodiment is not restricted to the disclosed methods of determining the distance between a micronucleus and a nucleus.  
      The above embodiments are to be understood as illustrative examples of the invention. Further embodiments of the invention are envisaged. It is to be understood that any feature described in relation to any one embodiment may be used alone, or in combination with other features described, and may also be used in combination with one or more features of any other of the embodiments, or any combination of any other of the embodiments. Furthermore, equivalents and modifications not described above may also be employed without departing from the scope of the invention, which is defined in the accompanying claims.