the term “ species ” as used herein with respect to nucleic acids refers to a group of nucleic acid molecules , all of which comprise the same nucleotide sequence , allowing all to hybridize under stringent conditions to the same probe . fig2 depicts basic steps in the overall microarray process 100 according to a preferred embodiment , and the form of the data produced and carried forward during this process . the data is stored in a computerized database system that has memory sufficient to hold the data collected from each step in the microarray process 100 , and has the capability of relating the data collected in each step with the other data collected throughout the process 100 . in the first step 200 of the microarray process 100 , the gene expression experiment is designed . in this step 200 , the genes to be tested are identified and recorded as data 202 for reference in any of the subsequent steps in the microarray process 100 . also , in this step 200 , the basic design aspects of the microarray process steps that follow this step 200 are designed . in the second step 204 , the microarray is fabricated . during this step , array fabrication data 206 is collected . fig3 depicts the preferred sub - steps performed in the array fabrication step 204 . these sub - steps include a microarray design sub - step 300 , a microarray setup sub - step 302 and a hybridization sub - step 304 . in the array design sub - step 300 , plate and array data 306 , 308 are gathered and used and the settings 310 for an arrayer that deposits the spots on the microarray slide are established . in sub - step 300 , the layout of the gene deposits on the slides is determined . certain parameters that describe the layout are also determined and recorded . these parameters include the number of trays , the number of slides per tray , the number of spots on each slide , the size of each spot expression region . the spot spacing and the pattern on the slides . in the arrayer setup sub - step 302 , the settings 310 for the arrayer are input and the microarray slides with spot samples are generated . in step 302 , the total number of plates , slides , etc . is determined and the microarray slides with samples are produced . furthermore , data on the laboratory conditions including the temperature , humidity , the airflow rate , the arrayer &# 39 ; s speed and acceleration , the operator and any other relevant parameters are recorded and saved . then , the hybridization sub - step 304 is performed and data 305 on the performance parameters for this step 304 , including the hybridization method . protocols and chemical buffers used and other experimental conditions are recorded and saved for use in one or more of the subsequent steps of the microarray process 100 . referring again to fig2 , after the array fabrication step 204 , an array - scanning step 208 is performed in which the array is scanned and a gray - scale digital image of the microarray slide is produced . in this step 208 , scanning parameters are also determined and saved as scanning data 210 that may be used in subsequent steps of the microarray process 100 . data for this step 208 includes , among other things , the gain , speed , pixel size , pattern and position size for the scanning device . once the microarray is scanned , in an image analysis step 212 , the resulting digital image is analyzed to extract the intensities at each sample location . image analysis data 214 is produced by this process step 212 and is used to perform , in a final step 216 , a gene expression analysis . fig4 illustrates a preferred system 400 for performing the microarray process steps depicted in fig2 and 3 . as depicted in fig4 , the system 400 includes a high - speed image processing computer 401 having a data input port 402 and output port 404 , a scanner 406 , and an arrayer 408 . the arrayer 408 places the spot samples on a glass slide 410 to generate a spot - filled microarray slide 412 . the arrayer 408 is typically comprised of an xyz cantilever robot holding 16 quill - pen type probes . the arrayer 408 also preferably includes a vacuum chuck for holding 48 standard microscope slides 410 , a microtiter tray loader / stacker , a wash / dry station , a controlling computer , air handling components , and a cabinet . the robot moves only the probe holder with the probes spaced on 9 mm centerlines to conform to the well spacing of standard microtiter trays . the microtiter tray / stacker / loader holds a plurality of microtiter trays and presents them one at a time to the robot load station when commanded by the controlling computer . the wash / dry load station flushes the probe tips with clean water and then dries them with a blast of clean air from the cabinet , the blast being pulled past the tips with the vacuum . the arrayer &# 39 ; s controlling computer controls all of the other component functions . once the arrayer 408 places the spots to generate the microarray slide 412 , the scanner 406 scans the microarray slide 412 and produces a digital image of the microarray slide 408 at its output 408 . the output of the scanner 406 is a digital image in a gray scale pixel format of the microarray 408 . the scanner 406 preferably includes a digital scanning sensor and an output port . the scanning sensor preferably further includes its own computing processor such as an intel ™ pentium ™ or another high - speed processor for controlling an inverted scanning fluorescent confocal microscope with a triple laser illumination system . the scanning sensor preferably performs at least 100 mm / sec scans with five - micron resolution . scanning is done in a comb pattern with data collected in both directions . the digital data is acquired using an integrator and preferably a standard 16 - bit a / d converter in the scanning sensor &# 39 ; s processor . once the digital image of the microarray is output by the scanner 406 , the image processing computer 401 processes the digital image 414 . the image processing computer 401 is preferably electrically connected at its input port 402 to the output port of the scanner 406 . the digital image 414 of the microarray slide 412 is then processed by the image processing computer 401 . preferably , the digital image 414 is processed in software operating on the computer 401 , the processing may be hard - wired , or it may be implemented in a combination of software and hardware without departing from the spirit and scope of the invention . at the output 404 of the computer 401 , the image analysis data 416 that is used to perform the gene expression analysis in the next microarray process step 216 is provided . this data generally includes measurements of the attributes that characterize the gene expression signal of each spot in the microarray . the basic steps for processing the microarray as a digital image in the processor 401 is shown in microarray processing sequence 500 of fig5 . while these basic steps and their sub - steps , as they are recited herein , are described in the context of microarray gene expression analysis , they may be applied to the analysis of other chemical ( including biological ) materials such as protein gels or tissues that are manifested as irregular spots or signals in an array of such spots or signals . the first step is the automatic detection of sub - grids 501 in the microarray image . a hypothetical example of a microarray image is shown in fig6 to conceptualize the automatic sub - grid detection step 501 . fig6 depicts a 2 . times . 2 “ meta - grid ” of four sub - grids . in the figure , each sub - grid has nine columns and ten rows . moreover , the meta - grid has two meta - columns and two meta - rows . in detecting the sub - grids , the rows and columns of each sub - grid of spots in the image are identified . the second basic step is circle localization 502 , wherein the center of each spot is identified . the third step is signal segmentation 504 , wherein the pixels in the microarray image that represent gene expression signal are identified . the fourth step is the calculation of quantity and quality measurements 506 , wherein features of the identified signal pixels and the background pixels are calculated and used to form the basis for , among other things , a confidence measure regarding the signal measurements . although each of the basic steps 501 , 502 , 504 , 506 is rigorously performed as later described , none of the basic steps assumes or relies on the perfect performance of any of the other basic steps . as such , the overall performance that is achieved in the microarray processing sequence 500 is superior to prior methods . fig7 depicts the steps performed in the automatic sub - grid detection process 501 . as discussed above , a microarray typically consists of a two - dimensional array of gene expression sub - grids . the first step 700 in the automatic sub - grid detection process 501 is to determine the general locations of the sub - grids in a microarray . specifically , preferably rectangular regions of a microarray are identified in which each identified region contains only one sub - grid . because the set of sub - grids in a microarray are normally configured as a two - dimensional array , the identification of each sub - grid region can be performed by identifying horizontal and vertical lines in the microarray that effectively isolate each sub - grid region . preferably , the method of identifying a horizontal or a vertical line that partitions a sub - grid region is the same whether the identified line is horizontal or vertical . as shown in fig8 , the method of identifying a horizontal or vertical line to isolate a sub - grid region is preferably comprised of the fourteen steps discussed below . the method is essentially the same regardless of whether a horizontal or vertical line is identified . outlining the steps for initially identifying a vertical line , the first step 800 is to sum all pixels in the microarray image in the vertical dimension to form an ordered one - dimensional horizontal vector . in the second step 802 , the maxima in the resulting one - dimensional vector are identified . to do this , a “ maximum ” filter is preferably used that returns a “ 1 ” if an element of the vector is the largest element in a window centered on the element . the window size is preferably set equal to the expected diameter in pixels of the spots in the microarray image . the diameter is preferably provided from the array scanning step 208 in the microarray process 100 and is normally between about 20 and about 30 pixels . similarly , in the next step 804 , the minima in the one - dimensional horizontal vector are identified . to identify the minima , a “ minimum ” filter is preferably used having a size that is the same as the maximum filter used in the previous step 802 . the minimum filter returns a “ 1 ” when the filter is centered on an element that is the smallest element in the filter window . once the locations of the maxima and minima are determined , in the next step 806 , the intensity differences between each maximum and each maximum &# 39 ; s nearest minima on each side in the ordered horizontal vector are calculated . by calculating the intensity difference , the relative height in intensity of each maximum is established . these relative heights are the “ peaks ” in the horizontal vector . next , a predetermined number , k , of the largest peaks are selected for consideration 808 . for the horizontal vector , k is preferably set to the number of meta - grid columns multiplied by the number of columns in a sub - grid . thus , k expectedly is equal to the total number of columns in the microarray . in fig6 , for example , k equals 18 . next , the mode of the distance between the selected k peaks is determined 810 . to determine the mode of the distance , the distance in pixels between adjacent peaks is found and then the mode for these distances is calculated . the mode distance value is used in the next step 812 to remove the weakest of the k peaks that were previously selected for consideration . this mode distance is used as the size of a maximum filter for pruning away the weakest of the selected peaks . for example , when the filter is centered on one peak , and another smaller peak is within the window of the filter , that smaller peak is removed from consideration . in the next step 814 , a process is initiated in which the remaining peaks are classified based on their heights as one of three kinds : valid , invalid or ambiguous . preferably , the classification step 814 begins by first , determining a first threshold intensity level for a valid peak . in a preferred embodiment , the first threshold level for a valid peak is equal to the median of the selected peaks multiplied by about 0 . 3 . in the second part of the step 814 , the peaks that exceed the first threshold level are classified as valid peaks . third , a second intensity threshold is determined to classify invalid peaks . the intensity threshold for invalid peaks is preferably set at the height of the lowest of the previously selected k peaks multiplied by 0 . 75 . in the fourth part of the classification step 814 , all of the peaks falling short of this second threshold are classified as invalid peaks . finally , the peaks below the first threshold but exceeding the second threshold are classified as ambiguous peaks . after completing this basic peak classification step 814 , the automatic sub - grid detection process 501 continues to the next step 816 , wherein peaks are inserted into the ordered horizontal vector . to perform the insertion , when the distance between two adjacent peaks is more than about 1 . 5 times the mode distance , then a peak is preferably inserted at the midpoint between them . the inserted peak is then classified as an invalid peak . this process step 816 is preferably performed for all of the originally selected k peaks . in the next step 818 , a score is computed for every set of c consecutive peaks , where c is equal to the expected number of columns in a sub - grid . the score for each set of c consecutive peaks is calculated by subtracting the number of invalid peaks from the number of valid peaks in each set . where the total number of peaks in the vector is n , there should be n − c + 1 overlapping sets of c consecutive peaks . once the scoring step 818 is performed , a process 820 for selecting for and eliminating from consideration sets of c consecutive peaks is initiated . first , the set of c consecutive peaks with the highest computed score is selected . once that set is selected , the sets of c consecutive peaks that overlap with this selected set are removed from consideration . this selection / elimination process 820 continues by selecting from the remaining sets the set of c consecutive peaks with the highest score , and then removing from consideration the sets that overlap the selected set . this selection / elimination process 820 is performed until all of the sets are either removed - or selected . in the end , it is expected that the number of selected sets equals the number of meta - columns in the microarray . in the next step 822 , the number of selected sets is compared to the number of meta - columns . if the number of selected sets does not equal the number of meta - columns , the automatic sub - grid detection process is considered unsuccessful . if automatic sub - grid detection process is considered unsuccessful , then the process to step 824 and exits . if the number of selected sets equals the number of meta - columns , the automatic sub - grid detection process is considered successful to that point and continues forward to step 826 . in this successful case , the selected sets of c consecutive peaks specify all of the columns of spots in the microarray where each set corresponds to the sets of columns for each sub - grid . according to the next step 826 , the previous steps in the sub - grid region detection process 700 are repeated . this time , however , the previous steps in the sub - grid region detection process 700 are performed to identify the meta - rows in the microarray image . finally , after all of the sets of rows and columns are determined , the microarray image is partitioned 828 into spatial regions according to the meta - row and meta - column - locations found in the above process steps . after the spatial region for each sub - grid has been partitioned , the automatic sub - grid detection process 501 proceeds to identify , as shown in fig7 , the rows and columns for each sub - grid of step 702 . again , the steps to find the rows in a sub - grid are preferably essentially the same as the steps for finding the columns . thus , only the process for identifying columns in a sub - grid is outlined below . fig9 depicts steps for a preferred row / column detection process 702 . in the first step 900 , all of the pixels in a sub - grid region along the vertical dimension are summed to form a one - dimensional horizontal vector . next , an “ averaging ” or low pass filter whose width is equal to the expected diameter of the spots is applied to the vector in step 902 . this averaging step 902 is performed because the image of each sub - grid region is smaller than the overall microarray image that was processed in the previous sub - grid region - locating step 700 . by applying the averaging filter , the noise that is inherent in a typical microarray image is reduced . next , the maxima or peaks in the horizontal vector are determined in step 904 , again using a maximum filter in which the size of the maximum filter is preferably equal to the expected spot size . in the next step 906 , using the previously calculated mode distance m to establish additional peak locations , peaks are added to the vector to fill the length of the sub - grid region . the resulting peak locations specify the locations of the columns in the sub - grid region . the previous steps for detecting the columns in a sub - grid region are repeated 908 to determine the locations of the rows in the sub - grid or vice versa . finally , a check step 910 is performed to determine whether the number of peaks for each vector is at least as high as expected . for the horizontal vectors , the number of peaks should equal or be greater than the expected number of columns in a sub - grid . for vertical vectors , the number of peaks should be equal to or greater than the number of rows in a sub - grid . if the number of peaks is less than expected for a horizontal or vertical vector , then the process exits at step 912 , having not performed successfully . if the number of peaks for a given vector is equal to or greater than the expected number , then the process exits at step 914 with the row and column detection process 702 being considered successful . with a successful completion of this process , the rows and columns define candidate sub - grids with grid - point intersections in each sub - grid region of the microarray . referring to fig7 , the next step 704 in the automatic sub - grid detection process 501 is the identification of a probable sub - grid in each region from the rows and columns identified in the previous step 702 . as discussed above , the identified number of rows and columns in each region should exceed the expected number of rows and columns of a sub - grid . thus , the specific rows and columns that correspond to the sub - grid in each region are next determined . fig1 outlines the sub - steps of step 704 in determining the probable sub - grid in each partitioned sub - grid region . first , a circular template is built in step 1000 , the circular template preferably having a circular center area of pixels with a ring of pixels around the center area of pixels . the diameter of the template is preferably equal to the shortest distance between grid points in a given sub - grid region . the size of the circular center area is preferably equal to the expected spot size . the pixel values in the circular center of the template are preferably set to “ 1 .” the pixel values in the ring around the circular center are preferably set such that the sum of all of the pixel values in the template is zero . thus , the pixel values in the ring region are all preferably set to some negative value , the magnitude of which depends on the ring size . in the next step 1002 , the filter is centered on each previously established grid point and a score is calculated . the score reflects the likelihood of a gene - expressing spot on that grid point . the score is computed by summing all of the pixels in the template window around the grid point after they are multiplied by the pixel weights in the circular template . in the next step 1004 , all of the grid points are rank - ordered according to their likelihood scores that were calculated in the previous step 1002 . next , new likelihood scores are provided for each grid point based on their rank order in step 1006 . the new rank order - based scores for each grid point preferably being determined by : ( 1 ) assuming that the sub - grid is expected to have r rows and c columns , for grid points having the top 0 . 5 . times . r . times . c likelihood scores , the new likelihood score is preferably set to 1 . 8 ; ( 2 ) for the remaining grid points that were in the top 0 . 8 . times . r . times . c likelihood scores , the new likelihood scores are preferably set to 1 . 0 ; ( 3 ) for the remaining grid points with likelihood scores among the top rc likelihood scores , the new likelihood scores are preferably set to 0 . 5 ; ( 4 ) all remaining grid points , their likelihood scores are preferably set to zero . in the next step 1008 , based on these newly assigned likelihood scores , candidate sets of sub - grids of dimension r . times . c are determined . sub - grid scores are determined for every possible sub - grid of size r . times . c in the sub region . the sub - grid score for a sub - grid is calculated in step 1010 by summing the likelihood scores of every grid point in the candidate sub - grid . then , the sub - grid with the highest likelihood score in each region is selected in step 1012 as the most likely sub - grid for that sub region . preferably , the next step 1014 is to determine whether the selected sub - grid is “ bounded .” a bounded sub - grid is one in which there are gene - expression spots in the leftmost column , the uppermost row , the rightmost column , and the lowermost row . if a sub - grid is unbounded on any one or all of its sides , then that sub - grid is considered “ free .” a determination of whether a sub - grid is bounded or free is made for the probable sub - grids in each region of the microarray . after the bounded - or - free determination is made for the probable sub - grids , the bounded sub - grids are used to constrain or “ bind ” any free sub - grids in constraining step ( s ) 1016 , 1018 . referring to fig7 , this step 706 is the last basic step in the automatic sub - grid detection process 501 . since the previous steps may not have confidently identified every probable sub - grid within the microarray , this constraining step 1016 , 1018 is performed . the purpose of this step 1016 , 1018 is to use the probable sub - grids that have been confidently identified to aid in fixing the locations of other probable sub - grids whose exact positions in their respective sub - grid regions remain uncertain ( i . e . are free ). for example , if a sub - grid is bounded on its left side and the sub - grid above it in the microarray meta - grid is unbounded on its left side , the position of the leftmost column in the lower sub - grid is used to constrain or “ bind ” the leftmost column of the upper sub - grid . the lowermost row , uppermost row or rightmost column of a bounded sub - grid is similarly used to constrain other free sub - grids . moreover , these constraints are preferably propagated from sub - grid to sub - grid as free sub - grids become bounded . after the constraint process has been exhausted to bind the free sub - grids , any remaining free sub - grids are constrained in a manner that minimizes the position offset between the free sub - grids and their neighboring bounded sub - grids . referring again to fig5 , after the automatic sub - grid detection process 501 has been completed , the next step 502 is circle localization . fig1 depicts the two basic steps in circle localization 502 . in a first step , an edge detection process 1100 is performed on the microarray image to generate an edgemap for the image . in the edge detection process 1100 , a number of different filters that are known in the art of image processing are optionally used . such filters include a sobol detector , a canny detector , a prewitt detector , a roberts detector , laplacian and gaussian methods and zero - cross methods . once the edge detection step is performed on the microarray image , and an edgemap has been created , the next step 1102 is circle template matching , an example of which is a hough transform . fig1 depicts a preferred embodiment of the specific steps in circle template matching process 1102 . the first three steps 1200 , 1202 , 1204 in the circle template matching process 1102 operate to identify the center pixel for a given gene - expression spot . to perform the spot center finding process , initially , a window around each grid point is established . the size of the window is the distance in pixels between rows by the distance in pixels between columns that were previously determined for the identified sub - grid . in this window around each grid point , every edgemap - pixel in the window is examined . for each pixel , the pixels orthogonal to the direction of the edge pixel and extending to the edge of the window are incremented by one in a counting array that maps to and has the same dimensions as the microarray window around the grid point . this incrementation of the counting array is the first step 1200 in the center pixel - finding process . by performing this operation on each pixel , a counter map is formed in step 1202 based on the incrementation of pixels in the window . after each pixel has been tested and the counter map has been generated , the maximum valued pixel in the counter map is identified in step 1204 . this maximum - valued pixel is considered to correspond to the center of the gene expression spot for that particular gird point . the next set of steps determines the radius of the spot around this determined center of the gene expression spot . in the first step 1206 of the radius finding process , the edge map pixels are pruned based on each pixel &# 39 ; s directional offset from each pixel &# 39 ; s direction to the identified center pixel . preferably , if an edge pixel is outside of a +/− 60 - degree angle window in its direction from the direction to the center pixel , then that edge pixel is removed from consideration . for all others that remain after the pruning step is completed , a histogram is formed in step 1208 based on the distance between each edge pixel and the center pixel . thus , the x - axis for this histogram is the distance of a given pixel from the previously identified center pixel . once this radius histogram has been formed the peak in the histogram is identified in step 1210 . this peak expectedly corresponds to a large number of edge pixels that are about the same distance from the identified center pixel . the location of this peak on the x - axis of the histogram is preferably established as the radius of the spot in step 1212 . by establishing the center and now the radius of the spot for a given grid point , the circle localization step 502 in the microarray process is completed . referring again to fig5 , the next step 504 is to segment or extract the gene expression signal from the region around each identified center pixel for each spot . fig1 depicts the basic steps in segmenting the signal using the identified center and radius defining a circle for each gene expression spot . the first step 1300 in segmenting the signal for a given spot region is to tentatively classify pixels inside of the circle as signal and to classify those outside of the circle as background . the next three steps 1302 , 1304 , 1306 in the signal segmentation process refine this tentative classification . the second step 1302 is to find the major mode in the intensity distributions of both the tentatively classified signal pixels and the tentatively classified background pixels . in fig1 , the performance of this step 1302 is expanded into eight sub - steps . the actual sub - steps in fig1 first outline the processing on the signal region . later , essentially the same steps are repeated on the background region . in fig1 , the first sub - step 1400 is to form an intensity histogram from the pixels inside the circle . next , the peak in the intensity histogram above the median intensity is identified in sub - step 1402 . having identified the peak , preferably all of the histogram bins with pixel numbers greater than the number of pixels in the peak bin multiplied by 0 . 7 are identified for consideration in sub - step 1404 . in the next sub - step 1406 , the identified histogram bins that are not part of a connected group of identified bins that includes the peak bin are removed . thus , after this sub - step 1406 , only a single group of connected bins should remain inconsideration . this cluster of bins that includes the peak bin is preferably identified as the major mode of the histogram . in the next sub - step 1408 , the slope on each side of the peak in the histogram is found . preferably , the following steps , which are essentially the same for the determination of both slopes , are performed to establish the slope on the high intensity side of the peak . first , the intensity histogram is differentiated and a differential histogram is created . the peak in the differential histogram is then identified , and then all of the differential histogram bins that are larger than 0 . 3 times the size of the differential peak are tentatively identified . the group of connected , tentatively identified bins that includes the differential peak is then identified . then , in the next step 1410 , from the group of identified bins , a slope line is fit on the original histogram . the same basic process of fitting a slope line is applied on the low intensity side of the peak in the original histogram to complete step 1410 . the two slope lines are finally extended to cross the x - axis on the original histogram . in the next sub - step 1412 , where the slope lines cross the x - axis of the histogram , the bins at these intersections are identified . an example of the result of the preceding operations on the histogram of tentative signal pixels is shown in fig1 a . in fig1 b , a similar histogram for the tentative background pixels is generated including the slope lines for that histogram . the slope lines for the tentative background pixel histogram are generated using the same sub - steps used to generate the slope lines in the tentative signal pixel histogram . thus , the same basic sub - steps in the major mode identification process 1302 are repeated for the background histogram in sub - step 1414 . the main difference in the process is that for the tentative background pixels , the histogram peak below the median intensity is initially identified rather than the peak above the median . in fig1 a , the bin at the intersection of the x - axis and the low intensity slope line of the peak in the signal histogram is identified as lsl for low signal level . the bin at the intersection of the x - axis and the high intensity slope line of the peak in the signal histogram is identified as hsl for high signal level . similarly , the corresponding intersections in the background histogram are lbl for low background level and hbl for high background level . this partitioning of the histogram is the next step 1304 shown in fig1 . by so the thresholding the histogram , pixels in the window are reclassified in the next step 1306 according to their spatial position and their position in the histogram , i . e ., whether they are in a group between lbl and hbl , between hbl and lsl , between lsl and hsl or above hsl . the classification of pixels according to their intensity and position with respect to the circle in the window is shown in fig1 a and 16b . specifically , in fig1 a , pixels with intensities above the hsl threshold are considered contamination pixels . pixels with intensities above the lsl threshold but below the hsl threshold , and that are inside of the circle are considered signal . pixels in the same histogram region but outside of the circle are considered “ undetermined .” pixels with intensities above the hbl threshold but below the lsl threshold and that are inside the circle are considered signal pixels . those outside of the circle with such intensity levels are considered “ undetermined .” finally , pixels with intensities below the hbl threshold are considered background pixels , regardless of their position with respect to the circle in the window . similarly , referring to fig1 b , pixels with intensity levels above the hsl threshold are considered contamination pixels . pixels with intensity levels above the hbl threshold and below the hsl threshold and that are inside the circle are considered signal pixels . pixels with such intensity levels that are outside the circle are considered “ undetermined .” finally , pixels with intensity levels below the hbl threshold level are considered background pixels . the distinction between pixels in the two figures is that , in fig1 a , the lsl threshold is greater than the hbl threshold , whereas in fig1 b , the hbl threshold is greater then the lsl threshold . finally , referring to fig1 , pixels that are undetermined are resolved in step 1308 . for these pixels , their positions in the window with respect to just - established signal and background regions is used to classify them . signal , background and undetermined pixels typically form somewhat homogeneous regions of each kind in the window . for example , a group of undetermined pixels may form an undetermined pixel region . similarly signal pixels often form a homogenous signal region . the spatial relationship of homogeneous regions to an undetermined pixel or pixel region is used to classify the pixel or pixel region as background or signal . in one preferred method of classifying an undetermined pixel region , if the region does not touch a signal region , the region is automatically classified as a contamination region . alternatively , if the undetermined pixel region touches a signal region but does not touch a contamination region , the undetermined region is classified as a signal region . as another alternative , if the undetermined pixel region touches both a signal region and a contamination region , the undetermined region is classified as one or the other depending on a further analysis . preferably , if the size of the contamination region that the undetermined region touches is smaller than ⅓ the size of the undetermined region , and the contamination region shares more than two - fifths of its border with a signal region , then the undetermined region is classified as a signal region . otherwise , the undetermined region is classified as a contamination region . optionally , this method of identifying spatial regions is repeated for any remaining undetermined regions . the newly identified spatial regions are used in the reperformance of this process . after all of the pixels have been classified , the spot in the segmentation window is identified by the pixels that have been classified as signal pixels in step 1310 . at this stage in the microarray image process , an analysis of the signal in the window is performed in step 506 . referring to fig5 , this step 506 is the last step in the microarray processing sequence 500 . in this step 506 , signal characterization and preferably , signal quality measurements are calculated based on the identified signal and background . the signal characterization measurements quantify the expression levels of the genes in different ways , while the signal quality measurements may be incorporated into confidence measure calculations associated with each gene expression signal &# 39 ; s measurements . signal characterization measurements include the mean of the intensity of the signal of a spot , the total number of pixels that comprise signal pixels ( signal area ), the median intensity and the mode intensity . the quality measurements are generally one of two kinds , local and global . local quality measurements are measurements in the window of a gene expression spot . these measurements include geometric properties of the signal and contamination regions in the window . one such measurement is the signal area referenced above . while being a signal characterization measure , signal area is also a signal quality measure . during array fabrication 204 , one of the goals of automation of the microarray process 100 is the achievement of consistency in the preparation of each spot sample . while expression levels from sample to sample may vary , ideally , each sample results in a gene expression signal that circular , centered on a grid point , of a certain radius , and thereby a certain area . the particular radius preferably depends on the parameters established during the experiment design and / or the array fabrication steps 200 , 204 . the measurement of signal area , and in particular , its deviation from a signal &# 39 ; s expected area as a result of the design of the overall microarray process 100 , is therefore a factor that becomes relevant to any determination of confidence in the signal &# 39 ; s measurement . another quality measurement that is preferably factored into any calculation of confidence is spot area . spot area differs from signal area in that spot area includes signal pixels and non - signal pixels that are located inside a predominantly signal region . fig1 a - b depict two examples distinguishing between spot area and signal area . in fig1 a , the signal area includes only the region labeled 1700 . the spot area includes both regions 1700 and 1702 . similarly , in fig1 b , the signal area includes only region 1704 , while the spot area includes both regions 1704 and 1706 . unlike fig1 a , in fig1 b , region 1706 is not completely surrounded by a signal region . region 1706 is , in part , defined by a segment 1708 that make the signal region appear more circular . the spot area , and additionally , its ratio relationship to the signal area is a signal quality measure that is potentially an indicator of spotting problems in the array fabrication step 204 . for example , a spot area that is significantly larger than the corresponding signal area may indicate a hole in the signal as shown in fig1 a that is often caused by problems with the shape of an arrayer pin , having an excessive amount of salt in the deposition solution , or problems with the chemistry of the slide . such issues are preferably factored into any confidence evaluation of a particular signal measurement while , at the same time , serving a quality control function for the overall microarray process 100 . another quality measure that is preferably factored into a confidence determination for the output measurements and preferably additionally serves as a quality control measure for the overall microarray process 100 is ellipticity or the degree to which the signal region has the shape of an ellipse versus a circle . to make this measurement , the length of the signal region &# 39 ; s major and minor axes are preferably determined . this measure both indicates the signal &# 39 ; s deviation from the desired circle and the potential that the signal &# 39 ; s elliptical shape is due to a problem in the array fabrication process 204 . for example , an elliptically shaped signal is potentially due to a directional airflow over the surface of the arrayer slide that causes the sample solution to spread in one or more directions on the slide surface . alternatively , an elliptical shape may be due to a slide that is not positioned horizontally or is uneven . to aid in narrowing the potential causes for an elliptically shaped signal and thereby refine the contribution to the confidence determination that is subsequently calculated , the orientation of the ellipse is also determined . in particular , by calculating elliptical signal orientation , the likelihood that directional airflow may be the cause of an elliptical signal can be more precisely determined . for example , if other signals on the slide have the same orientation , directional airflow becomes a more likely cause . conversely , if an elliptical signal &# 39 ; s orientation is an isolated occurrence , then the cause may be more likely a local anomaly such as an artifact on the slide surface . another geometric property measure that is preferably calculated is the square perimeter - to - spot area ratio . this measure is suggestive of a spot &# 39 ; s deviation from a circle and is preferably normalized to the measure &# 39 ; s value for a circle . because spots are preferably designed to be circular in shape , higher values for this measure preferably have a negative effect on the ultimate confidence values . the properties of contamination regions that may be present in a signal window are also preferably calculated and incorporated into the confidence calculation for the signal . one property is the area of the contamination region . larger areas of contamination preferably result in lower confidence values for an associated signal , and to some degree , the integrity of the signal generally may become questionable . another confidence measure is the difference in the average intensity for pixels in contamination regions versus the signal regions . this measure broadly indicates the confidence in the signal segmentation step 504 to distinguish between signal and contamination regions for a spot . smaller differences between the average intensities preferably result in lower confidences in their corresponding signal measurements . certain geometric properties of the sub - grids are also determined to support confidence measure determinations and provide further quality control indicators for the overall microarray process 100 . one such property is the deviation of a spot &# 39 ; s center location from its spot &# 39 ; s canonic location . this measure is calculated by determining for each spot the distance between its grid point established by the automatic sub - grid detection step 501 and the spot &# 39 ; s center , by calculating a centroid location for the spot , or optionally , using the spot center value determined in the circle localization step 502 . in confidence calculations for a signal measurement , larger differences preferably lower the confidence value to the extent that they indicate distortion in the spot placement , the existence of contamination , or an error in the performance of the automatic sub - grid detection step 501 . a quality measure that evaluates sub - grids more generally is degree of alignment between sub - grids ( sub - grid alignment ). this measure determines the degree to which a row or column of sub - grids are in alignment with each other . preferably , measures of sub - grid alignment are for pairs of sub - grids that are adjacent to each other either in the same row or the same column . for example , for two sub - grids in the same column , sub - grid alignment is preferably determined by first calculating the median of the distances between columns in both sub - grids . second , the median of the offsets between the corresponding columns in the sub - grids is calculated . finally , the sub - grid alignment value is determined by the ratio of the median of the distances between columns to the median of the offsets . the sub - grid alignment calculation is the same for adjacent sub - grids in the same row except that median calculations are applied to successive rows rather than successive columns . a determined sub - grid alignment value is potentially indicative of a pin printing error during the array fabrication step 204 , or may be indicative of the errors in the performance of the automatic sub - grid detection step 501 . for sub - grid alignment values greater than 0 . 5 , a problem in one of these two areas likely exists . for sub - grid alignment values greater than 0 . 3 , a closer inspection of the results of the process may be required . generally , however , the values are preferably factored into signal confidence determinations . another global quality measure associated with sub - grid analysis is the uniformity in the distance between sub - grids . sub - grid distance uniformity is a measure of the regularity of the distance between corresponding rows or between corresponding columns of two or more sub - grids . in a preferred embodiment , sub - grid distance uniformity is calculated by first determining the median of the distances between corresponding rows of adjacent sub - grids in the same column or corresponding columns of two adjacent sub - grids in the same row . a median distance value is therefore found for each pair of adjacent sub - grid in the microarray . the median of the set of median distance values for the pairs of sub - grids is then determined as a global median . finally , sub - grid distance uniformity is determined by the median distance value that has the largest absolute deviation from the global median . by calculating the sub - grid pair with the largest deviation , the calculation provides a clear warning of at least one sub - grid that is not in its expected location . like grid alignment , sub - grid distance uniformity is potentially indicative of pin printing problems such as a bent pin in the arrayer 408 or an error in the performance of the automatic sub - grid detection step 501 . furthermore , values for sub - grid distance uniformity are preferably factored into confidence measure determinations for measured signals . another set of quality measures concerns the variation in the identified background . locally , the background variation is the standard deviation in the intensity of background pixels for each window . higher standard deviations are potentially indicative of dust or other contamination on the local area of the window or possibly artifacts in the underlying glass or substrate . a measure of background variation is also determined for the entire microarray . to make this global determination of background variation , preferably the mean of the background for each signal window is first determined . then , the standard deviation of the accumulated background means is determined . a higher result for the standard deviation is indicative of variation or non - uniformity in large regions of the background . a likely cause of such non - uniformity is one or more large areas of contamination such as when fluorescent molecules on the slide outside a spot well become trapped between the slide and another plate , and are spread across the slide surface . another potential cause is an error during the scanning of the microarray into a digital image . the measure of global background variation is an indicator or such an occurrence and is preferably considered in any confidence calculations . another determination is whether any of the sub - grids in the digital image of the microarray appear to be missing . indications of one or more missing sub - grids suggest a contact problem associated with one or more pins of the arrayer 408 such as would be the case if a pin is bent or missing . the determination that a sub - grid is missing may also indicate an error in the automatic sub - grid detection step 501 . another signal quality measurement measures the parallelism of rows and of columns of sub - grids . such is measured by preferably calculating a slope for each sub - grid line in the microarray and then calculating the standard deviation of the slopes . this calculation is preferably performed for all rows and for all columns . similarly , the orthogonality between the rows and columns of the sub - grids is determined . orthogonality is preferably calculated by measuring the angle of the sub - grid lines at each sub - grid point intersection and determining the standard deviation of the measured angles . the measurement of both parallelism and orthogonality are indicative of potential errors in the automatic sub - grid detection step 501 or pin placement errors during array fabrication 204 . preferably , a set of monitoring calculations is performed as an additional quality control measure for the microarray process . the monitoring calculations include determining the intensity range and standard deviation for spots of the same known signal intensity that are inserted in the microarray image . furthermore , the microarray images are monitored by calibrating the intensity using a series of control spots at different intensity values and reporting the variances . the various signal quality measures , while being applied to identify potential concerns in prior steps of the overall microarray process 100 or in one of the prior steps of the image analysis process 212 , also preferably are combined into a function or system for determining a confidence value for each measured gene expression signal . the function may be one that is heuristically obtained based on the performance and analysis of previous results . alternatively , the various measures are included as input nodes to an artificial neural network that refines initial functional relationships between the signal quality measures . preferably , the confidence value is a percentage from 0 to 100 reflecting the system &# 39 ; s confidence in the obtained signal measurement . although the present invention has been described with reference to preferred embodiments , it will be readily appreciated to those of ordinary skill in the art that many modifications and adaptations of the invention are possible without departure from the spirit and scope of the invention as claimed hereinafter .