Abstract:
When reading a coupon that displays a pattern when a target substance is detected, recognition of the target pattern can be hindered by nonuniform illumination of the coupon. In one aspect, methods and devices are disclosed for uniform illumination of a coupon using a negative axicon lens and a light diffusing assembly. In another separate aspect, methods are disclosed for mathematically compensating for nonuniform illumination of a coupon.

Description:
RELATED APPLICATIONS 
       [0001]    This application claims the benefit of U.S. Provisional Application No. 62/322,357, filed Apr. 14, 2016, which is incorporated by reference as if fully set forth herein. 
     
    
     FIELD OF THE INVENTION 
       [0002]    The embodiments herein relate to devices and methods for reading a coupon that displays a pattern when a target substance is detected. 
       BACKGROUND 
       [0003]    A “coupon reader” is a device that accepts a “coupon,” typically a planar tray having a chemically reactive section that is imbued with a chemical mixture that is designed to react to a target substance and forms a visually detectable indicator pattern after a predetermined development time. A common implementation is the lateral flow immunoassay. The term “ticket” is also sometime used to refer to a coupon. Generally, coupons are designed to be read by a human technician, although coupon readers are also becoming common. The reader automates detection of the indicator pattern, removing reliance on human interpretation. Typically, a single company makes both coupons and the readers for those coupons, but there are also companies that do not make coupons, but do make coupon readers for coupons made by other companies (“third-party coupon readers”). Some coupons have only one section that reacts to a single target substance, whereas other coupons have a number of sections, each one designed to react to a different target substance. Coupons come in various sizes, but it appears that no currently available coupons are greater than 15 cm in any dimension. 
         [0004]    Coupons are used to detect substances of interest in the medical and public safety fields. For example, some coupons are designed to accept a bodily fluid onto the chemically reactive sections, to detect a naturally occurring target compound, such as a hormone, or a toxin. A public safety coupon can accept samples from many sources, including aerosolized particulates, liquids, and solids. Air-derived samples, mixed and concentrated into a liquid solution, and placed onto the chemically reactive sections, can provide an indication of an aerosol poison. Typically, the sample is incorporated into a buffer solution that is applied to the coupon, to facilitate the exposure of the coupon chemicals to the atmospheric agent or test specimen. Each chemically reactive section is referred to as a channel. Coupons may have multiple channels, each detecting a different target substance, allowing for multiple assays on a single coupon. Each channel typically includes a control section, which will develop in tandem with the test section of the coupon, but unlike the test section will display the indicator pattern, whether or not the target substance is present. The control section performs two functions, first, if the control section does not display the indicator pattern it is generally an indication that something has gone wrong with the process of exposure. Accordingly, a negative reading does not, in that instance, indicate an absence of the target substance, but only indicates a test that was not performed correctly. Also, it shows the test personnel what the indicator pattern looks like. 
         [0005]    In many situations, it is important to obtain a quick result from the coupon exposure, but the coupon manufacturer may have designated a set period of time for the indicator pattern to develop. Human technicians may set the coupon aside, and attend to other tasks while the coupon develops for the manufacturer specified time period. Variation in buffer solution and how it is applied can cause the same coupon to develop differently even when exposed to the same concentration of target substance. The use of a reader can lead to a faster detection of the indicator pattern when compared to a human, particularly in low light or stressful situations. 
         [0006]    For the purpose of analyzing the visually detectable indicator pattern of the coupon, it is desirable to provide uniform illumination over the surface of the coupon, as this allows full use of the camera&#39;s dynamic range over the entire field of view. As an example, if pixels directly under the light source have a reflected intensity of 255 units while more dimly illuminated pixels at the edge of the coupon have a maximum reflected intensity of 128, then the dynamic range and resolution of a measurement may be lower by a factor of two for assay channels near the coupon edge as compared to channels directly under the light source. In addition, if light rays illuminate a particular part of the coupon surface at oblique angles only, shadows can be created that confound analysis of assay results. These issues are compounded by the fact that a compact portable device is desirable. Most bioassay coupons are on the order of 11 cm in maximum diagonal measurement, and in the interests of having a compact device, it is desirable that the distance from the coupon to the camera lens is of similar magnitude. To maximize the number of pixels in the analysis, it is also essential that the coupon fill the field of view as much as possible. That is, a near-field macro imaging design is preferred. 
         [0007]    Light sources typically used for camera systems are so-called “white LEDs.” Uniform near-field illumination is sometimes accomplished using a circular array of white LEDs surrounding the camera lens, referred to as a ring light. Ring lights may have multiple circular rings of LEDs and may contain more than 100 LEDs. Such a large number of LEDs could significantly reduce battery life and thus operation lifetime in a battery-operated instrument. In addition, commercially-available ring lights still produce an illumination field in which intensity peaks in the center and drops off radially, albeit much more gradually than with a single LED. Since bioassay coupons are typically rectangular, ring lights may not provide uniform edge-to-edge illumination. 
         [0008]    Using a single LED light source is far preferable, but the hotspot problem must be solved. The LED cannot be mounted on the same axis as the camera lens: It must be physically located to the side of the lens, and the hotspot consequently appears at an off-axis camera image point. This does not improve the situation since parts of the coupon remain farther away than others from the LED, and light intensity over the coupon surface can be significantly nonuniform across the face of the coupon. 
         [0009]    Some improvement in light uniformity can be realized if a piece of ground glass or opalescent glass is mounted directly under the light source. This causes light to be re-emitted in a Lambertian pattern by virtue of the ground glass&#39;s surface roughness or by internal refraction in the opalescent glass. That is, the distribution of light emerging from the surface approximates a cosine function relative to a surface normal. These methods more broadly disperse the light, but there still remains a radial distribution of ever-decreasing light intensity relative to an axis passing through the LED source&#39;s center point and perpendicular to its surface. 
         [0010]    Therefore, there remains a need in the art for improved methods of reading a coupon that, among other things, minimize or compensate for variations in illumination over the surface of the coupon. 
       SUMMARY 
       [0011]    The teachings herein are directed to methods for reading a coupon that displays an indicator pattern when a target substance is detected, comprising providing a device having a computer readable memory, a digital camera, a logic assembly and a user interface; providing a target indicator pattern representative of an exposed and developed coupon; placing the target indicator pattern representation into the memory; exposing the coupon to a test fluid mixture; automatically repeatedly using the digital camera to take a digital image of the coupon after the exposure; after each of the digital images is taken, automatically using the logic assembly to compare it to the target indicator pattern representation to determine if it passes a similarity threshold; and in response to a digital image passing the similarity threshold, automatically providing a human perceptible indication that the target substance has been detected. 
         [0012]    Further embodiments are directed to methods for reading a coupon that displays an indicator pattern when a target substance is detected, comprising providing a device having a computer readable memory, a digital camera, a logic assembly and a user interface; providing a digitized target indicator pattern representative of an exposed and developed coupon, which is provided by taking a digital photograph of a coupon that has been exposed to the target substance after the predetermined development time has elapsed since exposure; placing the target indicator pattern representation into the memory; exposing an unused coupon to a test fluid mixture; automatically repeatedly using the digital camera to take a digital image of the coupon after the exposure; after each of the digital images is taken, automatically using the logic assembly to compare it to the target indicator pattern representation to determine if it passes a similarity threshold; and in response to a digital image passing the similarity threshold, automatically providing a human perceptible indication that the target substance has been detected. 
         [0013]    Additional embodiments are directed to methods for reading a coupon that displays an indicator pattern when a target substance is detected, comprising providing a device having a computer readable memory, a digital camera, a logic assembly and a user interface; wherein the device includes a substantially light tight enclosure which is lighted with an internal lighting assembly; providing a target indicator pattern representation representative of an exposed and developed coupon; placing the target indicator pattern representation into the memory; exposing the coupon to a test fluid mixture; placing the coupon into the enclosure and illuminating it with the internal lighting assembly, thereby illuminating the coupon in a manner that is repeatable between coupon exposures; automatically repeatedly using the digital camera to take a digital image of the coupon after the exposure; after each of the digital images is taken, automatically using the logic assembly to compare it to the target indicator pattern representation to determine if it passes a similarity threshold; and in response to a digital image passing the similarity threshold, automatically providing a human perceptible indication that the target substance has been detected. 
         [0014]    Additional embodiments are directed to methods for reading a coupon that displays an indicator pattern when a target substance is detected, comprising providing a device having a computer readable memory, a digital camera, a logic assembly and a user interface; providing a target indicator pattern representation representative of an exposed and developed coupon; placing the target indicator pattern representation into the memory; exposing the coupon to a test fluid mixture; automatically repeatedly using the digital camera to take a digital image of the coupon after the exposure; after each of the digital images is taken, automatically using the logic assembly to compare it to the target indicator pattern representation to determine if it passes a similarity threshold, including finding the coefficient of the target indicator pattern pixels that minimizes the sum of the squared differences between a nonlinear function of the target indicator pattern and the corresponding pixels of the digital image, and thresholding this coefficient to determine if a test indicator pattern of sufficient clarity has formed; and in response to a digital image passing the similarity threshold, automatically providing a human perceptible indication that the target substance has been detected. 
         [0015]    Still further embodiments are directed to an apparatus for reading coupons that display an indicator pattern after the passage of a predetermined development time after being exposed to a target substance, comprising a computer readable memory; a digital camera; a logic assembly, that controls the digital camera and interprets the data from the digital camera; a user interface; a user perceptible output; a substantially light tight chamber; a positional guide for placing one or more coupons into the substantially light tight chamber in predetermined and repeatable positions; and a lighting assembly for substantially evenly illuminating the predetermined and repeatable positions; and wherein the logic assembly controls the camera to repeatedly take an image of the coupon, after being user activated and after each of the digital images is taken, automatically comparing it to a target indicator pattern representation to determine if it passes a similarity threshold, and in response to a digital image passing the similarity threshold, automatically activating the user perceptible output. 
         [0016]    Additional embodiments are directed to an apparatus for reading coupons that display an indicator pattern after the passage of a predetermined development time after being exposed to a target substance, comprising a computer readable memory; a digital camera; a logic assembly, controlling and responsive to the digital camera; a user interface; a user perceptible output; a substantially light tight chamber; a positional guide for placing one or more coupons into the substantially light tight chamber in predetermined and repeatable positions; and a lighting assembly including at least one light source and a light diffusing assembly for substantially evenly illuminating the predetermined and repeatable positions, wherein substantially no light travels directly from the light source to the coupon, but all of the light illuminating the coupon has reflected off of a reflective surface on its path to the coupon; and wherein the logic assembly controls the camera to repeatedly take an image of the coupon, after being user activated and after each of the digital images is taken, automatically comparing it to a target indicator pattern representation to determine if it passes a similarity threshold, and in response to a digital image passing the similarity threshold, automatically activating the user perceptible output. 
         [0017]    Yet additional embodiments are directed to an apparatus for reading coupons that display an indicator pattern after the passage of a predetermined development time after being exposed to a target substance, comprising a computer readable memory; a digital camera; a logic assembly, controlling and responsive to the digital camera; a user interface; a user perceptible output; a substantially light tight chamber; a positional guide for placing one or more coupons into the substantially light tight chamber in predetermined and repeatable positions; and a lighting assembly including at least one light source and a light diffusing assembly for substantially evenly illuminating the predetermined and repeatable positions, wherein the at least one light source is substantially opposed to the positional guide, and wherein light from the light source is redirected by a negative axicon lens; and wherein the logic assembly controls the camera to repeatedly take an image of the coupon, after being user activated and after each of the digital images is taken, automatically comparing it to a target indicator pattern representation to determine if it passes a similarity threshold, and in response to a digital image passing the similarity threshold, automatically activating the user perceptible output. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0018]      FIG. 1  is a block diagram of a preferred embodiment. 
           [0019]      FIG. 2  is an illustration of common exposed coupon channel combinations of test and control pattern indications. 
           [0020]      FIG. 3  is a cross section of an apparatus for reading coupons according to one preferred embodiment, showing the position of the coupon relative to the apparatus and the path taken by light passing through the negative axicon lens. 
           [0021]      FIG. 4  is a graph showing the elimination of light intensity effects in a developed coupon pattern due to defining a ratiometric signal variable. 
           [0022]      FIG. 5  is a graph showing the application of least-squares nonlinear correlation to identify line strength and pixel location in a developed coupon pattern. 
       
    
    
     DETAILED DESCRIPTION 
       [0023]    Referring to  FIG. 1 , which is a block diagram of a preferred embodiment, in one preferred embodiment a coupon reader  10  includes a generally tight light chamber which is adapted to permit the introduction of any currently commercially available coupon (anything that is smaller than a 15 cm×10 cm×1 cm cuboid). The reader  10  includes a user interface  12  that prompts a user to enter the type of coupon being introduced into reader  10 . In one embodiment interface  12  includes a liquid crystal display coupled with a few keys, for making a choice of options presented on the display, and for providing a user perceptible indication. Interface  12  may also include a light or sound indicator for providing the human perceptible indication. In another embodiment, a voice recognition element is included. In yet another embodiment a USB port (not shown) is provided for a keyboard or a computer to be connected for commands and information to be sent to reader  10 . A computer readable memory  14  and a microcontroller  24 , driven by a clock  22 , collectively forms a logic assembly for controlling a digital camera  16  and interpreting digital imagery taken by camera  16 , to form a detection, or determine that no detection has occurred. A GPS receiver  20  determines location, which is input to microcontroller  24 . The location information together with test results is sent by way of a cellular network transceiver  18  over a cellular network link  28  to a remote station  30 , for storage. In one preferred embodiment, remote station  30  includes a mapping feature permitting users to see test results displayed on a map at the locations where the various test results were formed. 
         [0024]    In one embodiment, means are provided to introduce a target pattern representation (an image of a developed coupon) into a computer readable memory  14  of reader  10 . This data entry may be performed by placing a developed coupon or a control section into reader  10 , and using user interface  12  to command reader  10  to use a digital camera  16  to take a digital photograph of the developed coupon and store it in the computer readable memory  14 , properly labeled as a digitized image of a target pattern. In another preferred embodiment, reader  10  is provided with digitized target images already stored. Otherwise digitized target images may be introduced into reader  10  by way of the USB port {not shown}, or may be downloaded by way of a cellular network transceiver  18 . 
         [0025]    After an exposed coupon is introduced into reader  10 , the microcontroller  24  controls the digital camera  16  to repeatedly form images of each coupon channel and effectively compare each image with the digitized target image stored in computer readable memory  14 . 
         [0026]    Coupon manufacturers typically identify a specific time period that should elapse between exposing the coupon to the substance to be tested and reading the results on the coupon. In some cases, results may be visible before this predetermined development time has elapsed and time may be wasted by continuing to wait for the entire predetermined time period. The ability of the coupon reader  10  to determine when an indicator pattern has been detected and to alert any nearby people that detection has occurred helps to eliminate this wasted time. It will be appreciated that this ability is particularly important when the coupon reader is being used to detect the presence of toxic or harmful compounds in a public safety context, where time may be of the essence. 
         [0027]    Many other algorithms, including least squares detection and various linear algorithms are used in alternative embodiments.  FIG. 2  is an illustration of test patterns that are commonly encountered and which are amenable to interpretation and quantification using the mathematical and algorithmic approaches revealed herein. 
         [0028]    Prior to channel analysis, all embodiments require that the coupon image is recognized in the digital image being analyzed. Immediately following coupon image capture, image recognition routines analyze the captured image and identify channel locations based on the detection of characteristic coupon features. In embodiments, the user has entered the coupon type, and this information facilitates the recognition of the channel locations. The processing described below relates to each channel analyzed. 
         [0029]    Known optical features of the coupon are pre-loaded into the device before use. Image recognition tools identify the coordinates of these characteristic features for each different coupon type. The pixel coordinates of these features provide reference points and allow each coupon image to be overlaid by a coordinate map which can then be used to locate the assay channels. 
         [0030]    Upon positive recognition and satisfaction of location criteria, coupon analysis is allowed to continue. Upon detection of an abnormal situation, such as the coupon being inserted 90 or 180 degrees from the correct orientation, the user is alerted to the type of error. Once the error is remedied and location criteria are satisfied, sample analysis procedures may proceed. 
         [0031]    In one embodiment, each time a reader  10  processes a coupon, the results are time, position and device code stamped, with position being provided by a GPS reader  20  and time being provided by a clock  22  controlled by microcontroller  24 , and stored in computer readable memory  14 . In a preferred embodiment, this information is uploaded periodically via cell network link  28  using cell network transceiver  18 , to a remote station  30 . In one embodiment, remote station  30  includes a display and may be commanded to display a map of an area, showing the locations, times, results and device codes of each coupon reading. 
         [0032]    Referring to  FIG. 2 , illustration  1  shows a coupon channel in which neither test line T nor control line C developed. This could indicate an unused coupon channel, but could also indicate a coupon channel that has not been properly exposed to buffer solution. The absence of a control pattern indication, in this instance, prevents a false conclusion that the target substance is not present. Illustration  2  is a weak positive indication, due to the weak pattern. Illustration  3  is a strong positive, whereas illustration  4  shows an absence of the target substance. Illustration  5  indicates that something has gone wrong with the test, as the control line should always develop, especially if the test line appears. 
         [0033]    Referring now to  FIG. 3 , which is a cross section of an apparatus for illuminating and capturing coupon images, in one preferred embodiment the coupon reader  10  includes a light diffusing assembly  34  comprising side walls  36  having reflective interior surfaces  38  and a negative axicon lens  40 . The negative axicon lens  40  alters the path of light rays  42  emitted from light source  48  and passing thought the lens such that the light rays  42  reflect off the interior surfaces  38  prior to reaching the coupon surface  44 . The negative axicon lens  40  is designed to prevent any light from directly impinging on the coupon surface  44 . All LED output light is first operated on by the light diffusing assembly  34  before it impinges on the coupon. An optimum shape for the light diffusing assembly  34  is empirically determined, but it has been found that for illuminating a rectangular coupon, a concave structure that has a rectangular cross-section works well if the lower skirt of the structure approximates the coupon&#39;s outer shape. 
         [0034]    In one embodiment, the light diffusing assembly comprises side walls  36  which form a rectangular cross-section approximately 8 cm on a side. The side walls  36  meet the coupon surface  44  at angles that maximize uniformity of illumination over the coupon surface and compensate for the light source&#39;s off-center location. In a preferred embodiment, the included angle  46  between the side walls  36  and the coupon surface  44  is: 
         [0035]    Face  1 =70 degrees 
         [0036]    Face  2 =70 degrees 
         [0037]    Face  3 =70 degrees 
         [0038]    Face  4 =70 degrees 
         [0039]    In one embodiment, the distance between the coupon surface  44  and the negative axicon lens  40  is 15 cm. 
         [0040]    Diffuse reflection may be obtained from the reflective interior surfaces  38  by covering them with a flat or gloss white paint, or by constructing the structure using a white polymer. However, commercially available white polymers may absorb a significant amount of the optical power impinging on them, and this may need to be taken into consideration. 
         [0041]    Optionally, the reflective interior surfaces  38  may be covered with a colored coating rather than a white coating. A colored coating may be desirable in circumstances where a specific light spectrum distribution is preferred for illuminating certain types of coupon. In a further option, the reflective interior surfaces may be covered by a hybrid coating that provides a mixture of specular and diffuse reflection. 
         [0042]    Axicons are special lenses that have at least one active surface that is conic in shape. They will typically take a point source of light and transform it into either a line or circle of light. A negative axicon lens is used for coupon reader  10 . Typical commercially available axicon lenses are designed to form a specific geometric shape, and for that reason are considered “positive axicons.” The negative axicon, in contrast, takes a point of light and transforms it into an annular fan of light. Instead of a centrally located solid cone of material as in a positive axicon, the negative axicon has a conic pocket into which source light expands. 
         [0043]    Refraction at the conic top surface and at the optionally planar lower surface cause rays from a point source to be distributed over a range of angles determined by the conic profile, the distance of the source from the lens, the lens thickness, and the refractive index of the lens material. Angular emission of the lens can be tuned so that no light hits the coupon surface  44  without first reflecting off the reflective interior surfaces  38 . That is, in a preferred embodiment, no light passing through the negative axicon lens  40  falls directly on the coupon surface  44 , particularly at the spot immediately below the lens where a hotspot would normally occur. In this way, all light impinging on the coupon surface  38  is diffuse light re-emitted or reflected by the reflective interior surfaces and the uniformity of illumination over the coupon is greatly improved, even if the coupon is in close proximity to the light source and lens. 
         [0044]    It is not necessary that the conic profile of the negative axicon lens  40  be a simple cone. By adjusting the surface profile of the lens, the distribution of emitted optical power can be modified in an angular sense. Since the objective of the negative axicon lens and light diffusing assembly is to provide diffuse illumination, minor errors in the surface profiles are not critical. In one embodiment, the lenses are manufactured on a CNC lathe, followed by vapor polishing to remove macroscopic tooling marks. 
         [0045]    In one embodiment, a negative axicon lens with planar output surface for supplying optical power to the reflective interior surfaces is constructed of clear polycarbonate. In one embodiment, the negative axicon lens has the following dimensions: the overall height of the lens may be 3.5 mm, the diameter may be 10 mm, and the maximum depth of the cone may be 2.5 mm. 
         [0046]    In one embodiment, the cone profile is described as: Radius=1.98543z−0.18142z 2 +0.04361z 3 (where z is the distance in mm from the cone&#39;s vertex). 
         [0047]    In one embodiment, nonuniformity of illumination across the coupon surface is compensated for by color-based ratiometric analysis of pixel data. Most color cameras provide at least three color channels; typically red, green, and blue. It has been found that light intensity variations over a coupon due to geometric issues are very similar in the three color bands. Most lateral flow immunoassay coupons use colloidal gold as the line colorant and its peak absorption is typically in the wavelength range of 540 nm to 580 nm, which is principally apparent in the camera pixels sensitive to green and red. It has been discovered that by forming a pixel-by-pixel ratio of color channel intensities, a remarkable reduction in background nonuniformity is seen. For the common colloidal gold colorant, the red and green channels may be most useful in this regard, but creating a ratiometric intensity variable using other color channels may be preferred for other colorants. 
         [0048]      FIG. 4  shows raw intensity data for the green and red channels of a coupon imaged using a solid state white light flash illuminator. This coupon had previously been exposed to a liquid containing the target substance, and hence displayed the target pattern indicating a positive result. In this case, the target pattern constituted two colorimetric lines running the width of the channel. The coupon was imaged and the image pixels were divided into columns running parallel to the colorimetric lines. The values of the pixels in each column were summed to create the pixel data set. The large signal perturbation on the left side of  FIG. 4  exhibits the two absorbance maxima at approximate pixel locations  65  and  185 , respectively, due to colorimetric reactions on the test strip. The pixel signal minimum on the right, between pixel positions  400  and  500  is due to the control line and should always be present if the coupon is working properly. Note that there are significant intensity fluctuations in the baselines of both the red and green data sets. 
         [0049]    In one embodiment, a new variable is created when the green pixel intensities are divided by the corresponding red pixel intensities and the resultant curve is normalized to an arbitrary value of 1.0 (or 100%) at some arbitrary pixel location distant from the test and control lines. The resulting ratiometric variable is also shown in  FIG. 4 , normalized to a value of 100. Note the removal of spurious non-signal fluctuations seen in the green and red pixel backgrounds in sections away from the three absorbance maxima, caused by variations in the background light level. This allows the test and control lines to show up much more clearly as deviations from a near-constant ratiometric background signal. 
         [0050]    The fluid dynamic and chemical processes that create the test and control lines are typically identical or very similar. For most coupons, even those with multiple identification channels, all test and control lines produce pixel signal responses that are similar in shape and substantively differ only in overall intensity. Therefore, the relative concentration of a targeted material may be estimated by simply comparing the maximum deviation of the ratiometric signal from background and comparing it to the maximum deviation of the fully-formed control line. However, this may not be the most suitable method for test lines that have a small peak intensity because there may be too high of a signal to noise ratio to determine the peak value accurately. 
         [0051]    In addition, the test and target lines are of finite width and vary in intensity over the line&#39;s width. Therefore, the quantity of target material in the sample is more appropriately proportional to the integrated total response over the entire line width. Using the integrated response over the entire line width will also serve to smooth out pixel signal noise. For example, if a signal is averaged over 100 pixels, random background noise as a fraction of the mean signal value will typically be reduced by a factor of 10 times compared to the noise associated with a single pixel measurement process. 
         [0052]    In one embodiment, the following mathematical strategy and corresponding algorithm automatically provide a measure of the test and control line integrated areas and the pixel locations of each. The control and test line locations may indicate whether the assay coupon was correctly assembled or not, or if it was subsequently mishandled by the user. 
         [0053]    The coupon typically includes a control line, which develops even in the absence of the target substance to indicate that the coupon is functioning properly and that the correct procedures were used to expose and develop the coupon. In one embodiment, the control line is used as the source of reference information that permits the coupon reader to determine whether a test line is present or absent. In other embodiments, a different source of reference information may be used, such as a pre-printed line on the coupon, data from a different coupon, or an abstract representation of a developed control line, showing the expected result when the tested for substance is present. 
         [0054]    Because the test and control lines extend across the width of each coupon channel, the pixels are divided into columns running parallel to the test and control lines and then summed across each column. Each column sum defined in this way is identified by pixel position, and the value of the sum at that position is the pixel signal value for that position. The pixel signal values associated with a coupon&#39;s test and control lines can be approximated as the sum of a slowly varying background signal and a characteristically-shaped nonlinear absorbance signature, Y*(i), where “i” is pixel position. The aforementioned ratiometric signal between approximate pixel positions  400  and  500  in  FIG. 4  is such a characteristic control line signature Y*(i). In one embodiment, a two-variable nonlinear least squares regression technique may be used to test for the presence of signal lines with absorbance signatures similar to Y*(i). This is done by comparing test line data, Y(i), to a fitting equation, Y f (I), which is given as 
         [0000]        Y   f ( i )= a+b×i+c×Y *( i ) 
         [0055]    In this equation, “a” is the average baseline value, “b” is the rate of baseline change per pixel, and “c” is a measure of the goodness-of-fit of experimental data to the reference signature, Y*(i). To obtain an unbiased measure of this goodness-of-fit, we minimize the sum of squares of the deviations between the experimental data and the curve fit, Y f (i), 
         [0000]    
       
         
           
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         [0056]    The procedure determining the values of the parameters “a”, “b,” and “c” to minimize η are well-known in statistical mathematics. When this procedure is performed, it is found that the best-fit value of “c” is given by the following equation: 
         [0000]    
       
      
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         [0057]    The values K 1 , K 2 , and K 3  are constants that involve the reference signature Y*(i), and are easily calculated by someone knowledgeable in statistical mathematics. The other equation factors are calculated for each sectional pixel array of N+1 pixels length, [0, i, N], to be examined as follows: 
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         [0058]    The three summations are easily and quickly calculated and the constants K 1 , K 2 , and K 3  are only calculated once: After that they may be stored as look-up values for the channel. If the pixel data presented for comparison is the reference pixel array Y* i  itself, the value of “c” will be exactly 1.0 at the pixel location identical to the midpoint of the control line. 
         [0059]    As previously discussed, the test line in most cases is essentially identical in shape to the control line, but less intense overall. Hence, when the value of “c” is computed by scanning the test line section of the coupon channel pixel data with an array of [0, N] size, it will have maximum values when the signal data most closely matches the control line shape Y*(i). This allows identifying the location of each test line center point while the value of “c” represents the relative integral size of the discovered line as compared to the control line. For the example ratiometric pixel signal data in  FIG. 5 , it can be seen that the two test lines were robustly found and that the value of “c” for the control line is 1.0, as expected. 
         [0000]    This method is favored for several reasons:
       Small variations in the baseline are common due to lighting variations, shadows, and residual color in the coupon substrate. This method automatically estimates and corrects for a non-constant baseline. The integral value “c” is an unbiased best estimate of the baseline-corrected integral value.   Due to the use of all data points associated with a line, signal-to-noise is improved and a wide dynamic range is realized of as much as 100 to 1. That is, a test line with a value of “c” on the order of 0.01 may be detected.   The method allows development of a calibration curve that quantitatively correlates the targeted material&#39;s concentration with the test line integral value.   The method allows a quantitative determination that a test line&#39;s response is statistically above background noise.   The method provides the lines&#39; peak value location, which is useful in identification of manufacturing or user errors.   While the method can be applied to raw data as well as ratiometric data, the integral value from ratiometric data provides a measure of signal strength that is independent of absolute light intensity or light exposure time as long as the color channels all change proportionately the same when these factors are varied. This is a reasonable assumption for changes in exposure time or temperature, since both the control line and test line will see the same environmental shifts.       
 
         [0066]    In a preferred embodiment, for a multichannel coupon the three constants K 1 , K 2 , and K 3  are empirically determined for each individual channel by running one or more assays and calculating these least-squares constants for each channel&#39;s control line. This may be of particular value if there are differences in channel construction or if assay reagents are significantly different among the channels. 
         [0067]    In addition, in a multi-channel coupon the channel control lines may be compared one against another and these relative “c” values stored in the device&#39;s memory. During later use, the control lines can be compared to each other to see if any have deviated from the expected relative “c” values. The device may then issue a warning to the user that there may be a problem with one or more channels. 
         [0068]    In a preferred embodiment, digital images of the coupon are repeatedly taken over a period of time, starting shortly after addition of the sample and well before the time period suggested by the manufacturer has passed. These images are processed sequentially to determine if a test line has developed. As soon as detection positive result is found, a human perceivable signal is given. Depending on the coupon type, it is possible to gather useful information as early as 1 minute after development has been initiated. Some explosives coupons have a peak color at 2 minutes after application of a test fluid, and noticeably fade after 3 minutes. Accordingly, the time after exposure at which a first digital image is formed may be as little as 10 seconds or even 1 second. The techniques described herein have been shown to reduce the time to detection for bio-coupons to as little as 5 minutes, a third of the time period that the manufacturer recommends waiting, before evaluating an exposed coupon. 
         [0069]    Accordingly, in embodiments the taking of digital imagery begins well before the manufacturers development time has passed. In various embodiments, the taking of digital imagery begins at least 4, 3, 2 and 1 minute prior to the passing of the recommended time to develop, after exposure to said test fluid mixture. Described in different terms, in various embodiments a first digital image is formed no more than 1, 2, 3 and 4 minutes after the coupon channel is exposed to said test fluid mixture. 
         [0070]    In a further inventive feature, digital imagery is also repeatedly taken of the control section of each channel, beginning shortly after exposure to a test fluid mixture. This permits early detection of a coupon that has been ineffectively exposed to the test fluid mixture. As soon as ineffective exposure is determined, the test can be terminated and a new test begun, thereby greatly decreasing the time before an accurate reading is determined. 
         [0071]    In a further embodiment, the sequence of images are integrated together, to enhance the signal to noise ratio and, in some instances, provide earlier detection than would otherwise be possible. In one variant, time integration is performed on the raw pixels, but in another least squares fitting as described above is applied to the data from each digital image and the multiple results are then integrated over time. 
         [0072]    The invention may be embodied in other specific forms besides and beyond those described herein. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting, and the scope of the invention is defined and limited only by the appended claims and their equivalents, rather than by the foregoing description.