WO2001029756A2 - Autogrid analysis - Google Patents

Abstract

A method and apparatus for identifying the orientation and positions of grid elements in an image is provided. The grid elements do not need to be parallel to the sides of the image and the image does not contain all of the grid elements. The method can be applied to grids with several sides, straight or deformed. The addresses of the identified grid elements can be used to perform, for example, analytical procedures on individual elements.

Description

Autogrid Analysis

Background of the Invention Technical Field The present invention relates to methods of identifying objects in an image, and in particular, to identifying the orientation and position of a grid in an image, and using the resulting grid for measurement.

Description of the Related Art Many applications of machine vision and analysis analyze images which contain information arranged in a grid. For example, WO 99/08233 describes a large number of different types of analyses which can be conducted on assay well plates, gels and blots used in chemical, biochemical and physiological analyses. In that use, the positions of elements on a grid are identified and the elements are probed using a wide variety of assays.

WO 98/47006 takes this a step further, by using the image to identify locations of points on a grid, and injecting reagents onto the grid, e.g., with an ink jet sprayer. In both cases, however, the position and orientation of the grid are generally known. WO 99/08233 suggests allowing for some slight variation in the grid through the use of fuzzy logic, but the nature of the fuzzy logic to be applied is not described in the application. WO 98/47006 simply assumes one knows the orientation and size of the grid. Moreover, WO 99/53319 has recently suggested using shrinkable plastic. In this structure, the initial components of the grid are applied to plastic when it is large and the grid elements can be applied easily. The plastic then is shrunk, to reduce the size of the grid elements and resultant density of the elements being tested. This potentially makes manufacturing simpler and less expensive. However, such plastics may not shrink uniformly, so that the grids may be significantly distorted after shrinkage compared to grids on conventional glass or nylon substrates. Current image analysis systems are incapable of automatically identifying the positions of elements in such distorted arrays. In addition to these chemical applications, there are many manufactured products which include grid-like arrays or cross hatching, such as printed or micro-replicated materials, and chip sockets. Machine vision is a useful way of inspecting such products, but, again, there must be some ability to determine the orientation and position of each element in the grid for this to be useful.

In addition, most electronic analytical systems conduct measurements for every pixel in the field of view. In non-electronic viewing systems, there has been some suggestion to study only interesting features, e.g., to save the time for cytologists, GB

2,318,367 suggests having a system automatically identify interesting features and move a slide under a microscope to jump from one interesting feature to the next, so that the cytologist need only look at the interesting features. However, past conventional electronic scanning systems generally have scanned and analyzed every pixel in the field of view. Particularly if several measurements are being made, e.g., at different frequencies or under different excitation conditions, this collection of data from every pixel can be quite time consuming, since, for example, even a typical 512 x 512 pixel image has 262,144 pixels, and the scanning system must be physically re-adjusted on each pixel in turn. Summary of the Invention

The present invention identifies the orientation and position in a field of view of the elements or features of a grid. The grid may have a considerable degree of variance from a purely rectilinear grid.

To achieve this according to the present invention, an image of a field of view containing a grid is first obtained and digitized into pixels. Using any suitable algorithm, individual features in the image are identified. The centroid position of each feature, the feature area in pixels and the integrated intensity of the feature all are determined. These are used to produce a "collapsed image," where each feature is represented by a point object at the centroid location, with the point object having two associated values (area and integrated intensity).

According to a first embodiment of the invention, a search line is created at one side of the image at a base angle θ to the side of the image, and swept across the image in steps. At each step, the integrated intensity of each centroid within a predetermined region on either side of the line is determined and recorded. The result is a two dimensional plot with a series of peaks, each peak corresponding to a column of the grid. Note that this process is different from simply integrating the image intensity along the search line. Due to the use of collapsed images, each feature effectively has its image intensity and area concentrated at its centroid. This will be referred to herein as "centroid integration", and is discussed in more detail in a co-assigned U.S. Patent Application No. 09/422,584, filed on October 21, 1999, entitled "Centroid Integration.

Centroid integration results in a set of very well defined peaks in the resulting line profile. Regular integration would result in a set of smeared out peaks and, in the case of a grid with some variation in the positions of the individual features, the result would often be unusable. As a result, centroid integration is much more tolerant of local variation in feature positions than conventional integration.

Centroid integration is repeated with two additional search lines starting at a slight variance angle (+ δ and - δ) to the original search line and swept across the image.

The slope of the first peak on each of the three search lines is determined. The first peak represents the first column, and the steeper the slope on that peak, the closer that search line was to being parallel to the column of the grid. If the difference between the slopes of the three lines is above a predetermined threshold (i.e., outside a tolerance limit), the line with the steepest first peak slope is identified. If it is the middle line, the process is iterated using a smaller variance angle ±δ. If it is not the middle line, the base angle θ is reset to match the angle of line with the steepest slope, and the process is iterated using the same variance angle ±δ around the new base angle θ. The entire process is iterated until the difference is within the tolerance limit. The angle of the resulting line with the steepest first peak slope will match the angle of the first column of the grid (within the predetermined tolerance limit).

This process is repeated for each peak. For example, the second peak will correspond to the second column, so the search line with the steepest slope on the second peak is the closest to the angle of the second line of the grid. Repeating centroid integration using sweep lines at a base angle θ and variance angles (±δ) to find the slope of the second peak can define the angle of the second column. The process is repeated for each peak. After the first peak, it is not necessary to start the sweep lines at the side of the image - each sweep line can be started from position of the prior column. In addition, the angle of each column will probably be reasonably close to the angle of the prior column, so the number of sweeps usually can be minimized by starting with a base angle θ matching the angle of the prior column.

Once the position and orientation of each of the columns is identified, the next step is identifying the rows generally orthogonal to the columns just identified. The rows can be identified using substantially the same process as used to identify the columns, but starting from a side of the image adjacent to the side used to find the columns. The intersections of the best fit columns and the best fit rows are determined, and used to define the nominal points in the grid. The grid of nominal points preferably is "flexed" to define a final grid. To do this, a local search is performed at each nominal grid point for the local centroid, that is, the centroid nearest in position to the nominal grid point. The position of the local centroid is defined as the final grid point.

In some situations, only portions of a single object on a sample will appear in an image, with the results that multiple features are identified instead of a single feature. This might happen, for example, due to the characteristics of the object on the sample, or due to the image capture technique. An alternate aspect of the invention therefore is to find a centroid of the centroids within a predetermined distance of the nominal grid point, and to define this centroid of centroids as the final grid point. As will be apparent, if a single object is represented by multiple features, this will resolve the multiple features into an effective single feature, while if a single object is represented by a single feature, the centroid of centroids will be in the same position as the centroid of the single feature, so this will have no significant effect.

If no centroid or centroid of centroids is found within some predetermined maximum distance of a nominal grid point, it normally will be assumed that the feature (and centroid) expected at that point is missing, and the nominal grid point is retained as the final grid point.

The locations of all of the final grid points in the flexed grid provide an addressable array of points for the elements in the grid. According to a second embodiment of the invention, the corners of a grid array are identified first. To do this, a search line is started at a corner of the image, oriented with its normal bisector pointing towards the center of the image. The search line is moved towards the center of the image. The first centroid having an area and/or integrated intensity above a certain threshold limit which is encountered by the line is identified as the corner of the grid for that line. The remaining corners of the grid are identified in similar fashion with lines starting at the other corners of the image. The nominal outline of the grid array is defined by fitting a line between the four identified corners.

Next, a search is conducted along each of the lines in the nominal outline to identify additional centroids forming the outline. Preferably, this identification includes centroids within a predetermined distance of the nominal outline, to allow for a small amount of variability in the grid vertex positions.

If there are no features outside the nominal box plus a margin, then the grid is assumed to be (a) generally rectilinear. A search line is set up parallel to one of the lines forming the nominal outline of the grid. This search line is moved across the image in a series of steps to perform centroid integration, and identify the columns in the grid. A similar search line is set up parallel to an adjacent side of the nominal outline of the grid, and moved across the image to identify the rows in the grid.

If there are features outside one side of the nominal outline of the array, then the grid is assumed to be (b) curved toward that side. In the case of a grid which is distorted by curvature along one axis, a procedure similar to that for a rectilinear grid can be used to identify the grid points, but the procedure must take the curve into account. This is done by using a curved search line which matches the curvature of the grid. Any suitable method of identifying the curve can be used, and, once the curve is identified, the lines are moved in essentially the same fashion as with the rectilinear case (a) above.

In either the rectilinear or curved case, the intersections of the resulting best fit column and rows are used to define the nominal grid points. The grid is flexed to identify the local centroids and final grid positions.

The second embodiment generally requires the entire grid to be in the image, since it starts by finding the corners of the grid. In many situations, e.g., quality control of a large, moving web in production, the grid is not entirely within the image, but the orientation of the grid in the image is known, e.g., because the placement of the field of view relative to the web is known. If so, the grid spacing and position can be identified by using a variation of the second embodiment. In this variation, no attempt is made to identify the corners of the grid, since that is not possible. Instead of using search lines oriented parallel to the sides of the nominal outline defined by the corners of the grid, the same process as in the second embodiment is undertaken using search lines oriented roughly parallel to the expected columns and rows.

The third embodiment of the present invention can handle the most difficult situations, where the grid may be distorted in multiple directions. In this embodiment, the corners of the grid are found and a nominal outline for the grid first defined as in the second embodiment. The second embodiment assumes that the sides are generally a straight line, or at most curved in a single direction, but the third embodiment makes no such assumptions.

According to the third embodiment, after establishing the nominal outline, a search is conducted within a window on either side of one of the sides of the outline to find the centroids near that line. The distances between consecutive centroids along the line are calculated, and the histogram of distances is determined. The first large peak in the histogram corresponds to the nominal spacing along that edge.

The centroids making up the side (the first row or column) then are determined by an iterative process. Starting at one corner along the side, a search is made generally in the direction of the nominal side and a distance corresponding to the nominal spacing along that side. The closest centroid is found and identified as the next edge centroid. A new search then is made starting from the just-located centroid, generally in the direction of the line between the prior centroid and the just-located centroid and out a distance corresponding to the distance between the prior centroid point and the just-located centroid. This process iterates until the centroid at the other end of the side is reached. Having found the centroids forming the side, a best fit curve then is determined for those centroids. This curve is moved across the image to conduct centroid integration, and locate the centroids corresponding to the next row or column in the grid. If necessary, the centroids in this next row or column may be determined using the same process as described for the first row or column. However, it may be sufficient to simply generate a best fit curve of the centroids forming the centroid integration peak for the row or column. In either case, the new curve is used to find the next row or column. The process is repeated in the other direction, to find the columns or rows, and the grid is flexed, to find the actual grid centroids.

A fourth embodiment of the invention is useful for grids with other than four sides. In this embodiment, a first search line is moved in from the periphery of the image until it intersects a centroid. This centroid is initially assumed to be a comer of the grid. The search line then is pivoted around this comer centroid to perform centroid integration. The first peak found in the centroid integration defines the side ending at that comer. A search along that side identifies the centroid at the next comer. A new search line then is pivoted about the next comer to find the third comer. The process is repeated at each subsequent comer to find each side and comer until the original centroid is reached again.

It may be that a centroid identified by this technique is not actually a comer of the grid, but merely a centroid in a side significantly bowed outwards. One remedy to this situation is to assume that any comer for which the angle between the adjacent sides is greater than some pre-determined number, e.g., 65° for a hexagon which should have a nominal 60° comer angle, is not correctly identified as a comer. This remedy is useful if one knows the nominal shape of the grid. Another remedy requiring less prior information is to determine the angles formed at each comer, and graph them on a histogram. The peak in the histogram representing the smallest angle then is assumed to be the actual angle of the sides of the grid, and angles at least some amount (e.g., a few degrees or two standard deviations of the angles making up the largest angle peak) different than that are assumed to result from centroids which were initially, but erroneously, identified as comers. In either case, a nominal outline then is defined using only the correctly identified comers, and the grid points found through centroid integration as with any of the prior embodiments. Note that very little initial information is required about the number or spacing of the rows and columns in the grid, or about their orientation or position within the field of view. In the case of the first embodiment, all that is needed is to know that the grid is generally rectilinear. In the case of the second embodiment, all that is needed is to know that the grid is significantly curved in at most one direction and either that the entire grid is in the image, or that the orientation of the grid relative to the image is known. In the case of the third embodiment, all that is needed is to know that the image contains something that approximates a grid, even if heavily distorted. The fourth embodiment does not even require knowing how many sides the grid has. The method of the invention will identify all other information directly.

This low need for initial information has the distinct advantage of minimizing the care that need be taken in placing a grid in the field of view prior to imaging. For example, in the case of a grid used for combinatorial chemistry assays, the grid can simply be placed in the field of view for imaging, with no particular orientation or placement within the field required. Since accurate placement and orientation is time consuming, this can significantly accelerate review of large numbers of samples. The ability to handle even highly distorted grids allows for processing of materials with highly variable shapes, e.g., because the substrate they are made on is distorted during manufacture or use.

The foregoing analysis can be made from a single image of the field of view, without having to re-focus the system on each pixel. Further reductions in processing time can be achieved by using the information about the grid positions thus obtained to analyze just the significant locations in the field of view, not every pixel in the image. This can be done by re-adjusting the system to study just the pixels surrounding each centroid. Alternatively, the system can be re-adjusted so that the field of view substantially matches the feature around each centroid, and each feature can be analyzed in a single step. The system is re-adjusted from one feature to the next to analyze all of the features.

Typically, the number of features in a grid (e.g.., 96 in a DNA grid array) is significantly lower than the total number of pixels in the image (e.g., 262,144 in a 512 x 512 pixel image). Even if a moderate number of pixels (say 30 - 50) around each centroid must be analyzed, this dramatically reduces the total number of measurements (262,144 down to 2880 - 4800) which must be made and analyzed. If system is re-adjusted to match each feature, the total number of measurements needed is even lower, matching the number of features at 96. In either case, total processing time can be reduced markedly from the time required to measure every pixel in the image.

Brief Description of the Drawings Fig. 1 is a schematic illustration of a prototypical scanning system with which the present invention might be used. Fig. 2 is an illustration of an image which might be captured by the scanning system of Fig. 1.

Figs. 3a, 3b, 3c are illustrations of a scanning process applied to the image of Fig. 2 according to the present invention. Figs. 4a, 4b, 4c are graphs illustrating the initial results of the centroid process illustrated in Figs. 3a, 3b, 3c, respectively.

Fig. 5 illustrates a method according to the present invention of defining a set of nominal points on the grid in the image of Fig. 2.

Fig. 6 illustrates a method according to the present invention of defining a set of final points on the grid in the image of Fig. 2.

Fig. 7 is an illustration of a second image which might be captured by the scanning system of Fig. 1.

Fig. 8 illustrates a method according to the present invention for identifying the comers of the grid in the image of Fig. 7. Fig. 9 illustrates a method according to the present invention for identifying the nominal outline of the grid in the image of Fig. 7.

Fig. 10 illustrates a method according to the present invention for determining whether the grid in the image of Fig. 7 is likely to be rectilinear or curved.

Fig. 11 illustrates a centroid integration process applied to the image of Fig. 7 according to the present invention.

Fig. 12 illustrates a method according to the present invention of defining a set of nominal columns on the grid in the image of Fig. 7.

Fig. 13 illustrates a method according to the present invention of defining a set of nominal points on the grid in the image of Fig. 7. Fig. 14 illustrates a method according to the present invention of defining a set of nominal columns on the grid where the grid is curved.

Fig. 15 illustrates a method according to the present invention of defining the shape of the curve in the grid in the image of Fig. 14.

Fig. 16 illustrates a method according to the present invention of defining a set of nominal points on the grid in the image of Fig. 14. Fig. 17 is an illustration of a third image which might be captured by the scanning system of Fig. 1.

Fig. 18 is an illustration of a centroid integration process applied to the image of Fig. 17 according to the present invention. Fig. 19 illustrates a method according to the present invention of defining a set of nominal columns on the grid in the image of Fig. 17.

Fig. 20 illustrates the position of pixels around an individual grid feature in any of the images of Figs. 2, 7, 14 or 17.

Fig. 21 illustrates a process of re-adjusting the system of Fig. 1 to encompass an entire feature in any of the images of Figs 2, 7, 14 or 17.

Fig. 22 is a detailed illustration of features representing a particular object in the image of Fig. 2.

Figs. 23a, 23b illustrate a method according to the present invention for determining the distances between centroids forming a possibly distorted grid. Fig. 24 is a histogram of the distances determined in Fig. 23b.

Fig. 25 illustrates a method of according to the present invention for identifying the actual centroids forming a row or column in a grid.

Fig. 26 illustrates a highly distorted grid mapped according to the present invention. Figs. 27-30 illustrate an embodiment of a method according the present invention which is particularly useful with grids having other than four sides.

Detailed Description of the Preferred Embodiments Fig. 1 illustrates a scanning system with which the present invention might be used. In the system of Fig. 1, a focused beam of light moves across an object and the system detects the resultant reflected or fluorescent light. To do this, light from a light source 10 is focused through source optics 12 and deflected by mirror 14 onto the object, shown here as a sample 3x4 DNA array plate 16. The light from the light source 10 can be directed to different locations on the sample by changing the position of the mirror 14 using motor 24. Light that fluoresces or is reflected from sample 16 returns to detection optics 18 via mirror 15, which typically is a half silvered mirror. Alternatively, the light source can be applied centrally, and the emitted or fluoresced light can be detected from the side of the system, as shown in US 5,900,949, or the light source can be applied from the side of the system and the emitted or fluoresced light can be detected centrally, or any other similar variation. Light passing through detection optics 18 is detected using any suitable image capture system 20, such as a television camera, CCD, laser reflective system, photomultiplier tube, avalanche photodiode, photodiodes or single photon counting modules, the output from which is provided to a computer 22 programmed for analysis and to control the overall system. Computer 22 typically will include a central processing unit for executing programs and systems such as RAM, hard drives or the like for data storage. It will be understood that this description is for exemplary purposes only; the present invention can be used equally well with "simulated" images generated from magnetic or tactile sensors, not just with light-based images, and with any object to be examined, not just sample 16.

Fig. 2 illustrates an image of the field of view captured by any suitable system, such as that show in Fig. 1. If not initially created in digital form by the detecting equipment, the image is digitized into pixels. Features of interest in the image are identified by any suitable means. For example, features that are brighter or darker than a certain threshold intensity or features that are between two limiting threshold intensities might be identified as features of interest. Individual features are composed of touching pixels that satisfy the image intensity threshold requirements. This feature detection ability is available in most commercial image capture software packages. A detailed description of one method for achieving this can be found at The Image Processing Handbook, second edition by John C. Russ (CRC Press 1995) pages 394-96, 416-18.

Once the features are identified, a "collapsed image" is created. To do this, the centroid of each feature is determined, that is, the point that represents the center of the object based on a weighting scheme. A given feature may or may not be circular, so any suitable algorithm for identifying the centroid may be used, such as integrating intensity or position. The position of the centroid may be recorded in any suitable coordinate system, but typically will be in an X-Y coordinate system. In addition to the centroid, the feature area in pixels, and the integrated intensity of the feature all are determined, and stored in the memory of computer 22. The resulting collapsed image condenses the enormous amount of data in the complete image, e.g., 512 x 512 pixels of information, to a much smaller array, e.g., 3 x 4 x 4 for sample 16, which nevertheless still contains the information needed for present purposes, resulting in dramatically improved processing times.

Note that while the invention is described herein in terms of lines and curves moving across an image, with centroids being within predetermined distances of lines or points, and of graphical analysis of the results of such actions, this is for ease of description. It will be understood by one of skill in the art that it is not necessary to actually make such steps visible on a screen or other display device. Computer 22 can make the relevant calculations, or any mathematically equivalent calculations, without providing a display in the process. For example, computer 22 can process the data most expeditiously by comparing the locations and values in the condensed image array to the calculated locations of the relevant lines and points to which the centroids are being compared.

Also, as used herein, "column" will be used to indicate general alignment of the centroids of a grid in one direction, and "row" to indicate general alignment of the centroids in a direction generally orthogonal to the columns. It will be understood that which direction is the column and which the row is entirely arbitrary, so no significance should be attached to the use of one term over the other, and that the rows and columns may not be entirely straight. Referring to Fig. 3b, in a first embodiment according to the invention, search line

300 is created at one side of the image at a base angle θ to the side of the image (θ can be 0, as shown in the drawing), and swept across the image in steps. At each step, the integrated intensity of each centroid within a predetermined region (the "smoothing window") on either side of the line is determined and recorded. The result is a two dimensional plot with a series of peaks, each peak corresponding to a column of the grid, as shown in Fig. 4b.

This process is different from simply integrating the image intensity along the search line. Due to the use of collapsed images, each feature effectively has its image intensity and area concentrated at its centroid. This will be referred to herein as "centroid integration". With proper selection of the size of the smoothing window, centroid integration results in a set of very well defined peaks in the resulting line profile. Regular integration would result in a set of smeared out peaks and, in the case of a grid with some variation in the positions of the individual features, the result would often be unusable. As a result, centroid integration is much more tolerant of local variation in feature positions than conventional integration.

A smoothing window W is used to take into account the local variability of the centroids from their nominal positions. The window size should be based on the pitch of the rows and columns being analyzed. The desired size of smoothing window W is that size which will most clearly generate distinct peaks in the integrated intensity during centroid integration. The smoothing window can be adjusted manually or by any technique which can establish distinct peaks. The minimum value for W in pixels is 1. If the pitch spacing P in pixels is known and is greater than 4, the optimum smoothing window W in pixels will be about: W = 0.35 * P- 1.53

This equation has been determined empirically by evaluating a wide range of samples with local feature variations ranging from 0 (no deviation from a regular grid) to 50% (the variation in feature position is so large that the rows or columns overlap).

If the pitch spacing P is not known, one technique for automating the initial selection is to assume that the number of features in each row and column is the square root of the number of features identified in creating the collapsed image, then to approximate the pitch P of the rows and columns by dividing the size of the image by the square root of the number of features. Alternatively, if the nominal dimensions of the grid can be determined (see the second embodiment below), the pitch P can be approximated by dividing the nominal grid dimensions by the square root of the number of features. In either case, the initial smoothing window W can be determined using the equation above. If needed, this initial smoothing window W can be optimized by re-evaluating data to the data using a smoothing window W' varying around this smoothing window W, and selecting the smoothing window W' that provides the clearest peaks. Turning to Fig. 3 a, centroid integration is repeated with a second search line 302 at a slight variance angle (+δ) to the original search angle θ, and in Fig. 3c, with a third search line 304 at a slight variance angle (-δ) to the original search angle θ.

The slope of the first peak 400, 402, 404 (Figs. 4b, 4a, 4c, respectively) in the resulting centroid integration for the three search lines 300, 302, 304 is determined. The first peak in each graph represents the first column in the grid, and the steeper the slope of the first peak, the closer that corresponding search line was to being parallel to the column of the grid.

If the difference between the slopes of the three peaks 400, 402, 404 is above a predetermined threshold (i.e., outside a tolerance limit), the line 300, 302, 304 with the steepest corresponding first peak slope is identified (line 304 in the example shown in the drawings), and the process is iterated using varying values of θ and δ, until the differences are within the tolerance limit. To this, if the peak 400 has the steepest slope, the next iteration preferably uses the same value for θ as the prior iteration, but a smaller value for δ, while if the peak 400 does not have the steepest slope, the next iteration preferably uses the same value for δ as the prior iteration, but resets θ to match the angle of whichever of lines 302, 304 corresponds to the peak 402, 404 with the steepest slope. The entire process is iterated until the difference between the three slopes is within the tolerance limit. The angle of the final line with the steepest first peak slope will match the angle of the first column of the grid (within the predetermined tolerance limit).

A best fit match at that angle is made of centroids within a predetermined distance (which may or may not be same as the smoothing window W) of the final line to define the nominal position of the first column.

While the best fit for the columns can be identified by finding the crest of the peaks in the centroid integration, columns alternatively may be identified by using the blank regions between peaks in the centroid integration plot to determine the limits on each column, particularly if the blank regions are easier to identify in a particular situation. The centroids within each defined column are used to define a best fit line for the column, with each centroid being weighed by a combination of its area and/or its integrated intensity. This process is repeated for each peak. For example, the second peaks 406, 408, 410, correspond to the second column, so the search line with the steepest slope on the second peak is the closest to the angle of the second line of the grid. Repeating centroid integration using sweep lines at a base angle θ and variance angles (±δ) to find the slope of the second peak can define the angle of the best fit line for the second column. The process is repeated for each peak.

After the first peak, it is not necessary to start the sweep lines at the side of the image. Each sweep line can be started from the position of the prior column, as shown by line 312 in Fig. 3b. In addition, the angle of each column will probably be reasonably close to the angle of the prior column, so the number of sweeps usually can be minimized by starting with a base angle θ matching the angle of the prior column.

Once the position and orientation of each of the columns is identified, the next step is identifying the rows generally orthogonal to the columns just identified. The rows can be found using substantially the same process just used to identify the columns, but starting from a side of the image adjacent to the side used to find the columns.

The intersections of the best fit columns and the best fit rows are determined, and used to define the nominal points in the grid, as shown in Fig. 5. Preferably, to avoid singularities in the fitting, the equations describing the columns are of the form x = my+b (as opposed to the conventional y = mx+b used for the rows). The nominal grid of Fig. 5 is "flexed" to match the actual centroid locations to the nominal locations to generate a final grid, as shown in Fig. 6. This is done by performing a search for a local centroid within a predefined distance from each nominal grid point. If a local centroid is found, then the position of the local centroid is designated as the final grid point. In some situations, only portions of a single object on a sample will appear in an image, as shown in Fig. 22, which is a close-up view of the image of Fig. 2 around a single nominal grid point 2220. This results in multiple features 2200 being identified instead of a single feature. This might happen, for example, due to the characteristics of the object on the sample, or due to the image capture technique. To overcome this possibility, preferably the centroids 2210 of the features 2200 which are within the predefined distance of the nominal grid point 2220 are identified. A centroid 2230 of these centroids 2210 determined, and the position of this centroid 2230 of centroids 2210 then is defined as the final grid point.

If no local centroid or centroid of centroids is found, then the location of the nominal grid point is used as the position of the final grid point, on the assumption that a feature in the grid is missing.

After flexing, the result is an array of addresses representing the positions of the actual or expected elements of the grid, which can be used for further analysis, as described below.

While the foregoing first embodiment can accommodate grids that are rotated and grids that are distorted such that the grid elements are still generally in straight lines, it has difficulties with grids that are distorted into curved lines. A second embodiment therefore is provided which can be used instead of or in addition to the first embodiment. The second embodiment is effective for grids that are curved in a single direction, as sometimes results from shrinkage of a polymeric or other substrate along a particular axis. Referring to Fig. 7, an image is captured. The significant features in the image are identified and a collapsed image created in the same fashion as in the first embodiment. In Fig. 8, computer 22 creates search line 801 at one comer of the image, with its normal bisector pointing toward the center of the image. Computer 22 steps search line 801 toward the center of the image. The centroid of the first feature which has an area and/or integrated intensity above a predetermined threshold which is encountered by search line 801 as it moves toward the center of the image is identified as the comer centroid for that search line. This process is repeated with search lines 802, 803, 804 from each of the other comers. Referring to Fig. 9, lines 900, 902, 904, 906 are defined between the comer centroids just identified to define the nominal outline of the grid. It will be appreciated that while the lines have been shown and described as moving from the comers of the image, this is not necessary; the search lines could move from any edge of the image. In addition, if confidence is high in the dimensions of the grid, two approximately orthogonal search lines would be sufficient to identify the orientation and placement of the grid. In Fig. 10, computer 22 searches outside of the nominal outline of the grid for additional centroids. Centroids within some predetermined margin of the nominal outline will be considered "within" the outline, to allow for some local position variation in the grid, but centroids beyond that margin are considered outside of the nominal outline. If all of the centroids are within the nominal outline of the grid, then it can be assumed that the grid is (a) generally rectilinear. If centroids fall outside of the nominal outline, then it is likely that the grid is (b) curved in at least one direction.

If the grid is considered to be (a) generally rectilinear, the process of the first embodiment can be used to identify the columns and lines. Alternatively, in Fig. 11, computer 22 starts a search line parallel to a side of the nominal outline of the grid, and moves it toward the opposite side, performing centroid integration along the way. A best fit of the centroid integration peaks is made to define the best fit columns shown in Fig. 12. Essentially the same process is repeated in the opposite direction of the grid to find the nominal rows. The nominal positions of the grid elements are defined by finding the intersections of the best fit columns and rows, as shown in Fig. 13, and the grid points flexed, as described in the first embodiment. If the grid is curved, as shown in Fig. 14, upon checking for centroids outside of the nominal grid outline, centroids will be found to be outside of the nominal outline, so it will be assumed that the grid is (b) curved. If centroids are found outside just one side of the nominal outline or it is otherwise is known that the grid is likely to be curved only in a single direction, then the simplest method for identifying the nominal grid positions is shown in Figs. 14-16.

In this method, it is assumed that the curve is convex on the side towards which the centroids were found outside of the nominal outline. The sides adjacent to that side are presumed to be generally straight. A process like that used in either the first embodiment or in Figs. 11 and 12 can be used to identify the columns in this array parallel to the generally straight sides, as shown in Fig. 14. Once the columns are identified, a search down each column starting from the end thereof either towards or away from the convex side can quickly identify the first centroid in each column, which presumably forms a portion of the first curved row. A best fit is done of the first feature from every column to create a nominal curve, as shown in Fig. 15. The nominal curve is moved down the grid to perform centroid integration, but using a curve instead of a line. The result, shown in Fig. 16, is a nominal grid of straight columns and curved rows. As in the other embodiments, the intersections of the nominal columns and curved rows define the nominal grid points, and the grid is flexed to identify the final grid points. In many applications, particularly those involving a moving web during manufacturing, only a portion of the grid may be visible in the image. The process of the first embodiment can handle this situation directly, but the complete process of the second embodiment cannot, since no comers can be identified. However, in this situation, it normally can be assumed that the image orientation can be predetermined by virtue of the positioning of the image detector on the web production line, so that the grid orientation in the image is known, such as shown in Fig. 17. In this situation, the expected grid orientation can be used to establish the orientation of the search line. A search line can be started parallel to the expected orientation, as shown in Fig. 18, and moved across the image performing centroid integration. This will result in peaks of integrated intensity that can be used to generate best fit lines, such as shown in Fig. 19. The process can be repeated in the opposite direction, resulting in a set of nominal best fit lines in that direction. If the grid is curved, the process just described for the second embodiment can be used to define the needed curve. Intersections of the resulting rows and columns define a nominal grid, which can be flexed to identify the final grid points.

As will be apparent, the present invention can accept grids in many orientations. Given this flexibility, it is generally advisable to provide some characteristic element or feature on the grid which identifies a specific side or comer of the grid. For example, one feature in the grid array could be used as a marker. Then, no matter what the orientation of the grid in the field of view, the correct orientation of the grid elements relative to predetermined information, such as the nature of the chemicals being testing in each grid element, will be known.

Once the position, orientation and dimensions of the grid are known, this information can advantageously be used to reduce the time required to collect the information actually being measured. In a typical image, a given feature will span several pixels. As shown in Fig. 20, feature 2000 spans a 6 x 6 set of pixels 2010. To scan the pixels in feature 2000, motor 24 moves mirror 14 to direct the scanning beam on each pixel 2010 in turn. Measurements are made of each pixel 2010. Alternatively, as shown in Fig. 21, the scanning beam can be re-adjusted to have a diameter 2110 slightly larger than the feature 2100. Using this technique, the entire feature can be measured at one time. This is particularly advantageous when measuring the total luminescence or similar characteristic of a feature. The scanning beam can be re- adjusted on each feature in the grid. The total number of measurements needed thus is minimized, and exactly matches the total number of significant features.

The first steps of the third embodiment are conducted in substantially the same manner as the first steps of the second embodiment. Search lines are established and moved toward the center of the image until they intersect a centroid. The four centroids found this way are assumed to be the comers of the grid, and a nominal outline of the grid is established between them. However, the second embodiment assumes that the gird is probably generally straight, or at most curved in a single direction, but the third embodiment makes no such assumptions.

Having determined the comers and nominal outline of the grid, the third embodiment proceeds to determine the nominal spacing between the centroids forming the side of the grid. Referring to Fig. 23a, starting at the centroid 2301 at one end of the side 2303 of the nominal outline, a search is conducted for centroids within a window defined by margins 2304, 2305 on either side of the side 2303. The distance between each centroid and the next centroid within the window along the side is determined, as shown in Fig. 23b. The value of the first peak 2307 in a histogram of these distances, shown in Fig. 24, then is assumed to be the nominal spacing between centroids along the side 2303.

Ideally, the size of the margins 2304, 2305 is one half of the nominal distance, but since that is not known at the outset, some approximation must be used. A simple initial approximation is to divide the length of the nominal side 2303 by the square root of the number of centroids in the image.

Note that the presence of any peaks 2308 in the histogram beyond the first peak 2307 may indicate that the side 2303 is so curved that a point (such as 2309 in Fig. 23a) is outside of the search window, or that the initial approximation for the size of the search window was too small, e.g., because the grid is not a square, and the initial approximation therefore was highly inaccurate. In some circumstances, e.g., if there are quite a number of peaks in the histogram, it may be desirable to iterate this process using a new search window defined using one half of the nominal distance found on the first iteration to define the margins 2304, 2305.

Once the nominal spacing is defined, the third embodiment then finds each point in the side 2303. As shown in Fig. 25, a search is conducted starting from centroid 2301 at one end of the nominal side 2303. The search looks for the centroid closest to the nominal distance away from the centroid 2301, and generally in the direction toward the opposite end of the nominal side 2303. This closest centroid 2501 then is assumed to be the next centroid on the side 2303. The process then is repeated using the distance from centroid 2301 to centroid 2501 instead of the nominal distance, and searching generally in the direction of the line between centroid 2301 and centroid 2501, instead of in the direction of the nominal side. The process then iterates until reaching the centroid 2502 at the opposite end of nominal side 2303.

Having found the centroids actually making up the side of the grid, a best fit, e.g., a third order polynomial, is made of those centroids to define a curve. This curve then is moved across the image to the opposite side of the grid to perform centroid integration, just as the line was moved in the second embodiment. The position of the first centroid integration peak after the side is assumed to be approximate position of the next row or column in the grid.

While it is possible to define the subsequent grid rows or columns by finding the points closest to this initial curve, if the grid distortion varies a great deal, this will be a poor fit. Therefore, it is may be necessary to repeat the process used to find the centroids forming the side at each row or column in the grid, but using the curve established by the prior grid as the base line instead of the line used for the nominal side. A new curve then is established for each row or column. However, in many situations it will be sufficient to establish the new curve by making a best fit of the centroids forming the centroid integration peak for the row or column being analyzed, which is much simpler. In either case, the new curve is used to search for the next row or column.

This process then is repeated starting from a nominal side roughly orthogonal to the nominal side first used. The intersections of all of the defined curves then are used to define the nominal grid, and the grid is flexed, just as in the prior embodiments.

Preferably, when the grid is flexed the local search for a centroid near to each nominal grid point is conducted over an area based on the spacing of the grid around that nominal grid point, as determined in the course of defining the rows and columns above, e.g., over a radius one-half of the distance between the point and its nearest neighbors. Since grid distortion may vary considerably on a local scale, this will adapt the search to the local scale and any distortions in it.

The result of this is an accurate grid mapping even for highly distorted grids, such as that shown in Fig. 26.

A fourth embodiment of the invention provides a further refinement which is particularly useful with a grid having more or less than four sides. For purposes of description, a grid 2700 which nominally has six sides is shown in Fig. 27, one side 2701 of which is somewhat distorted. A search line 2703 is swept in from one side of the image until it intersects a centroid 2704. This first centroid 2704 is assumed to be a comer of the grid 2700.

Referring to Fig. 28, a search line 2800 then is pivoted about the first centroid 2704 to perform centroid integration. The first peak in the centroid integration will occur at the angle corresponding to the side 2801. A search then is conducted along the direction of this angle from the centroid 2704 to locate the centroid 2804 at the other end of the same side (much as was done along each side in the third embodiment above), which is assumed to be the next comer. Referring to Fig. 29, a search line 2901 then is pivoted about the second centroid

2804 to perform centroid integration. Preferably, the line 2901 starts positioned in line with side 2801, then sweeps toward the center of the image.

As noted before, the side 2701 is distorted. This means that centroid integration along the search line 2901 will identify a false side ending in a false comer centroid 2902 that is not actually a full side and comer of the grid. Nevertheless, for the moment centroid 2902 is treated as the next comer, a new search line 2904 is pivoted around it, to find yet another false side and false comer centroid 2905. The process is repeated with search line 2906 to find centroid 2907, and then repeated all the way around the grid until centroid 2704 is again reached. The comers (both real and false) thus identified are then used to define a nominal outline 3000, as shown in Fig. 30. As will be apparent from the figure, the angles 3001 at the real comers will be smaller than the angles 3002 at the false comers. This can be used to distinguish between the two types of comers to identify the real comers. If the nominally correct angle is known, e.g., 60° for the hexagon shown, then any angle greater than 60° (or perhaps, 65° to allow for some distortion) can be assumed to be a false comer. Alternatively, a histogram of all of the angles can be prepared. The peak representing the smallest angle (and having a value greater than 1 , to avoid problems from a highly distorted comer) then identifies the nominally correct angle. Any angles not forming part of that peak then represent false comers. The width of the peak can be defined arbitrarily, e.g., a few degrees, or calculated based on the data, e.g., determine the standard deviation of the angles making up the peak, and consider any co er having an associated angle more than two standard deviations away from that to be a false comer. In either case, the false comers then are omitted, and a new nominal outline 3003 defined. Note that with this process, it does not matter if the first centroid 2704 encountered by the initial search line 2703 is a real or a false co er - the process proceeds in the same manner either way. Once the nominal outline is defined, the locations of the centroids in the grid 2700 then can be defined using any of the techniques described in the other embodiments.

As will be apparent, different approaches described above can be combined in different ways. For example, when conducting centroid integration to identify a row or curve in the grid, the third embodiment calls for use of the curve generated for the prior row or column, but that curve, or variants of it, can be swept across the image to find the best fit, as discussed in connection with Fig. 3 above. Or, if the position of the nominal outline is known from some other source, e.g., if a user identifies the comers of the grid in the image, it would not be necessary to find the comers of the grid at the beginning of the second and third embodiments. The order in which many of the steps of the different embodiments of the invention are executed also may be varied from that described and claimed here, while still achieving the same result and without departing from the scope of the present invention.

It therefore will be understood that these exemplary embodiments in no way limit the scope of the invention. Other modifications of the invention will be apparent to those skilled in the art in view of the foregoing description. These descriptions are intended to provide specific examples of embodiments which clearly disclose the present invention. Accordingly, the invention is not limited to the described embodiments or to the use of specific elements, dimensions, materials or configurations contained therein. All alternative modifications and variations of the present invention which fall within the spirit and scope of the appended claims are covered.

Claims

Claims We claim:

1. A fluorogenic compound of the formula:

wherein:

Q is a enzymatically hydrolyzable group selected from the group consisting of a glycone, a glycosyl phosphate, an ester, and a peptide; each R² independently is a sterically non-interfering group;

R³ is an electron withdrawing or non-electron withdrawing group;

Z is O or NR⁵, wherein R⁵ is hydrogen or a hydrocarbyl-containing group;

Y and Y¹ independently are O, S, NH_X, or CH_y where x is 0 or 1 and y is 1 or 2, and at least one of Y and Y¹ is O, S, or NH_X; and each R⁴ independently is selected from the group consisting of hydrogen, halogen, a C]-C₂o alkyl, a Cι-C₂₀ alkoxy, a C₃-Cι₈ cycloalkyl, a C₆-Cι₈ aryl, a C₆-Cι₈ aryloxy, a C₆- Cι₈ hydroxyaryl, a C₆-Cι₈ arylcarboxy, a C -Cι₈ carboxyaryl, a C₂-C|₈ alkenyl, a Cι-C₂o hydrocarbylamino, a C -Cι₈ arylamino, a C₆-Cι₈ aminoaryl, a C₂-C ₀ di(hydrocarbyl)amino, a heterocyclic group having at least three ring atoms, carboxyl, carboxamide, ester, keto-alkyl ester, sulfonic acid, amino, and a group of the formula (CH₂A)_aE in which A is O, NH, or a single bond, E is a functional group that includes active hydrogen, and a is a whole number from 1 to 100; or both R⁴ groups together with the carbon atoms to which they are attached form a 5- or 6-membered aromatic ring which optionally can have one or more R⁴ groups attached; or a salt thereof.

2. A compound according to claim 1, wherein: each R² independently is selected from the group consisting of: hydrogen, halogen, a Cι-C₂o alkyl, a Cι-C₂₀ alkoxy, a C₂-C₁₈ alkenyl, a Cι-C₂o hydrocarbylamino, a C₂-C₂₀ di(hydrocarbyl)amino, and a group having the formula (CH₂A)_aE in which A is O, NH, or a single bond, E is a functional group that includes active hydrogen, and a is a whole number from 1 to 100; and

R³ is selected from the group consisting of: hydrogen, a C C o alkyl, a C₃-Cι₈ cycloalkyl, a C₆-Cι₈ aryl, a C -Cι₈ aryloxy, a C₆-Cι₈ hydroxyaryl, a C₆-Cι₈ arylcarboxy, a C₆-Cι₈ carboxyaryl, a C₂-Cj₈ alkenyl, a C -Cj₈ alkynyl, a halogen selected from the group consisting of F, CI, Br, and I, a Cι-C₄ haloalkyl, a cyano, a C]-C cyanoalkyl, a carboxylate, a C]-C₆ carboxylalkyl, and a group having the formula (CH₂A)_aE in which A and E are defined as above and a is a whole number from 1 to 100.

3. A compound according to claim 1, wherein: each R² independently is selected from the group consisting of: hydrogen, halogen, a Cj-Cio alkyl, a Cι-C₎₀ alkoxy, a C₂-Cι₀ alkenyl, a Cι-C*o alkylamino, a CpCio dialkylamino, and a group having the formula (CH₂A)_aE in which A is O, NH, or a single bond, E is a functional group that includes active hydrogen, and a is a whole number from 1 to 100; and R is selected from the group consisting of: hydrogen, a Ci-Cio alkyl, a C₅-C₈ cycloalkyl, a C₆-Cιo aryl, a heterocyclic group comprising at least one O, N or S atom, a C₂-Cιo alkenyl, chloromethyl, a C₂-C₎₀ alkynyl, a halogen selected from the group consisting of F, CI, and Br, a Cι-C haloalkyl, a cyano, a Cι-C₆ cyanoalkyl, a carboxylate, a Cι-C carboxylalkyl, and a group having the formula (CH₂A)_aE in which A and E are defined as above and a is a whole number from 1 to 25.

4. A compound according to claim 1, wherein Z is O or NR⁵, where R⁵ is hydrogen or a Ci to C₄ alkyl group.

5. A compound according to claim 1, wherein each R⁴ independently is selected from the group consisting of hydrogen, halogen, carboxyl, carboxamide, ester, keto-alkyl ester, sulfonic acid, amino, and a group of the formula (CH₂A)_aE in which A is

O, NH, or a single bond, E is a functional group that includes active hydrogen, and a is a whole number from 1 to 25; or both R⁴ groups together with the carbon atoms to which they are attached form a 6-membered aromatic ring which optionally has one or two R⁴ groups attached.

6. A compound according to claim 1, wherein: Z is O; each R² and R³ are hydrogen; and each R⁴ independently is selected from the group consisting of hydrogen, halogen, carboxyl, carboxamide, ester, keto-alkyl ester, sulfonic acid, amino, and a group of the formula (CH₂A)_aE in which A is O, NH, or a single bond, E is a functional group that includes active hydrogen, and a is a whole number from 1 to 10; or both R⁴ groups together with the carbon atoms to which they are attached form a 6-membered aromatic ring which optionally has one or two R⁴ groups attached.

7. A compound according to claim 1 , wherein Q is selected from the group consisting of α- and β-D-galactopyranosyl; α- and β-D glucopyranosyl; N-acetyl-α- and β

-D-galactosaminyl; N-acetyl-α- and β-glucosaminyl; β-D-glucuronyl; α-L- arabinopyranosyl; α-L-arabinofuranosyl; β-D-fucopyranosyl; α- and β-L-fucopyranosyl; α-D-mannopyranosyl; β-D-xylopyranosyl; α-D-maltosyl; β-D-lactopyranosyl; β-D- cellobiosyl; α-d-N-acetylneuraminyl; and myoinositol-1-yl phosphate.

8. A compound according to claim 1, wherein Q is α- and β-D- galactopyranosyl, α- and β-D-glucopyranosyl, or β-D-glucurosyl.

9. An enzyme sensing composite structure comprising: a support; and a fluorogenic compound according to claim 1 , wherein the compound is covalently bound to the support through at least one R², R³ or R⁴ by means of one of a bond and a linking group, said linking group comprising functionalities at both ends, the functionality at one end of said linking group being complementary to the functionality of R², R³ or R⁴ and the functionality at the other end being complementary to a functional group on said support.

10. The composite structure of claim 9, wherein the linking group is hydrophihc .

11. A fluorogenic macromolecular conjugate comprising: a ligand selected from the group consisting of a molecular ligand and a macromolecular ligand; and a fluorogenic compound according to claim 1 , wherein the compound is covalently bound to the ligand through at least one R², R³ or R⁴ by means of one of a bond and a linking group, said linking group comprising functionalities at both ends, the functionality at one end of said linking group being complementary to the functionality of R², R³ or R⁴ and the functionality at the other end being complementary to a functional group on said ligand.

12. The conjugate of claim 11 , wherein the functionalities of the linking moiety independently are an amine, amide, ester, oxirane, olefin, urea, silanol, carbamate, isocyanate, thioisocyanate, sulfonamide, sulfonyl chloride, sulfonic acid, carboxylic acid, carboxyl, chlorotriazine, hydrazine, hydrazide, or aldehyde.

13. The conjugate of claim 11, wherein the ligand is selected from the group consisting of a protein, a polypeptide, a glycoprotein, a carbohydrate, a steroid, a non- biological organic compound, and a non-biological organic polymer, and combinations thereof.

14. A method of determining the effectiveness of a sterilization procedure, comprising the steps of:

(a) placing a test indicator containing an active enzyme in a detectable concentration into a sterilization chamber,

(b) performing the sterilization procedure within the chamber;

(c) introducing a fluorogenic compound of claim 1 and allowing or providing a means for enzyme to diffuse such that the fluorogenic compound is hydrolyzed by the enzyme to form a cleaved compound, whereby the cleaved compound, when exposed to light of a wavelength range centered around ^SE_J , is capable of emitting light of a wavelength centered around ^SE₂, wherein ^SE₂ is at least 10 nm greater than ^SE_I, ^SE_I is at least 380 nm and ^SE₂ is no more than about 700 nm; and

(d) exciting the cleaved compound with light of a wavelength range centered around for a time sufficient for the cleaved compound to emit visible light of wavelength ^SE₂; and

(e) detecting and analyzing the emitted light.

15. The method of claim 14, wherein the enzyme is selected from the group consisting of β-D-glucosidase, α-D-glucosidase, alkaline phosphatase, acid phosphatase, butyrate esterase, caprylate esterase lipase, myristate lipase, leucine aminopeptidase, valine aminopeptidase, chymotrypsin, phosphohydrolase, α-D-galactosidase, β-D- galactosidase, α-L-arabinofuranosidase, β-D-glucuronidase, N-acetyl-β-glucosaminidase, β-D-cellobiosidase, alanine aminopeptidase, proline aminopeptidase, tyrosine aminopeptidase, leucine aminopeptidase, phenylalanine aminopeptidase and a fatty acid esterase derived from spore-forming microorganisms.

16. The method of claim 14, wherein the test indicator containing an active enzyme in a detectable concentration is arrayed in a discontinuous pattern on a support.

17. A method of detecting a biological target molecule in a test sample, the method comprising the steps of:

(a) providing a fluorogenic macromolecular conjugate of claim 11 wherein the ligand is identical to the biological target molecule; (b) incubating the test sample with a predetermined amount of the fluorogenic macromolecular conjugate and a predetermined amount of a specific binding partner for the target molecule, wherein the target molecule and the fluorogenic macromolecular conjugate compete for binding by the specific binding partner, further wherein the predetermined amount of the fluorogenic macromolecular conjugate is chosen such that a significant fraction of the fluorogenic macromolecular conjugate becomes bound and rendered inaccessible to enzymatic hydrolysis;

(c) adding to the test sample an enzyme that will enzymatically hydrolyze the unbound fluorogenic macromolecular conjugateto form a cleaved fluorogenic compound, whereby the cleaved fluorogenic compound, when exposed to light of a wavelength range centered around λi, is capable of emitting light of a wavelength centered around λ₂, wherein λ₂ is at least 10 nm greater than λ_{] ;} λi is at least 380 nm and λ₂ is no more than about 700 nm; and

(d) exciting the cleaved fluorogenic compound with a light of a wavelength range centered around λi for a time sufficient for the cleaved compound to emit visible light of wavelength λ₂; and

(e) detecting and analyzing the emitted light

18. An enzyme sensing element comprising:

( 1 ) one or more fluorogenic compounds of claim 1 ; (2) a fluid handling architecure structured and adapted to provide mixing of one or more enzyme-containing samples with at least one of the fluorogenic compounds so as to enable an enzymatic reaction wherein a cleaved fluorescent product is formed, such that the cleaved fluorogenic compound, when exposed to light of a wavelength range centered around λi, emits light of a wavelength λ₂, wherein λ₂ is at least 10 nm greater than λi, wherein λj is at least 380 nm and λ is no greater than about 700 nm.

19. The sensing element of claim 18, wherein the fluid handling architecture is selected from the group consisting of a test card, a microwell array, a capillary array, a microfluidic chip, a sensor disk, an array of sensor disks, and combinations thereof.

20. The sensing element of claim 18, wherein the fluid handling architecture is configured to absorb a fluid sample containing viable microorganisms and to support the growth of the viable microorganisms in microcolonies.