Patent Publication Number: US-8532348-B2

Title: Iterative processing

Description:
CROSS REFERENCE TO RELATED APPLICATION 
     This application claims the benefit of U.S. Provisional Application No. 61/107,988, filed Oct. 23, 2008, which is incorporated herein by reference. 
    
    
     FIELD 
     The disclosure pertains to the analysis of chemical and biological specimens. 
     BACKGROUND 
     In an experiment, peaks found on images formed from a liquid-chromatography/mass-spectrometry process are evidence that illuminates pharmaceutical discovery. For one image, a bounding area may be defined to localize a peak, which represents a biological clue that is of interest. For the remaining images, there may be many other peaks, all representing potentially different biological clues of interest. However, the same peak must be identified and localized in the remaining images. Furthermore, to reduce the risk of including undesirable artifacts in the peak bounding area, which could lead to erroneous scientific conclusions, the localized bounding areas across all images should be the minimum bounding area required to encompass the peak across all images. 
     SUMMARY 
     This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This summary is not intended to identify key features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter. 
     One aspect of the present subject matter includes an iterative regional processor, which comprises a regional redefiner configured to execute on hardware to alter a bounding area for peaks that point to a species across constituent images so as to facilitate quantitation of the peaks, the bounding area being used to localize peaks across constituent images. As used herein, a peak is a variation in an analysis signal due to a specimen under investigation. In combined liquid chromotagraphy (LC)/mass spectrometry (MS), such peaks are typically associated with an elution time (or time interval) and a mass to charge (m/z) ratio. Examples of peaks include a single peak, a cluster of peaks, or other contiguous signal variations. 
     Another aspect of the present subject matter includes a method for processing regions of interest to discover biological features, which comprises selecting whether to use a composite image to redefine a region of interest or a set of constituent images to redefine the region of interest without using the composite image. The method further comprises redefining the region of interest to alter a bounding area for peaks that point to a species across constituent images so as to facilitate quantitation of the peaks, the bounding area being used to localize peaks across constituent images. 
     A further aspect of the present subject matter includes a computer-readable medium having computer-executable instructions stored thereon for implementing a method for processing regions of interest to discover biological features, which comprises selecting whether to use a composite image to redefine a region of interest or a set of constituent images to redefine the region of interest without using the composite image. The method further comprises redefining the region of interest to alter a bounding area for peaks that point to a species across constituent images so as to facilitate quantitation of the peaks, the bounding area being used to localize peaks across constituent images. 
     The foregoing and other objects, features, and advantages of the invention will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The foregoing aspects and many of the attendant advantages of this invention will become more readily appreciated as the same become better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings, wherein: 
         FIG. 1A  is a block diagram illustrating exemplary hardware components to process prepared biological samples to produce biological candidate lists to researchers. 
         FIG. 1B  is a block diagram illustrating exemplary hardware components to execute a iterative regional redefinition process. 
         FIGS. 2A-2C  are pictorial diagrams illustrating exemplary LC/MS images (constituent images). 
         FIG. 2D  is a pictorial diagram illustrating an exemplary composite image. 
         FIGS. 3A-3B  are pictorial diagrams illustrating snippets of constituent images and exemplary microaligned snippets. 
         FIG. 3C  is a pictorial diagram illustrating an exemplary composite snippet. 
         FIG. 4A  is a pictorial diagram illustrating an exemplary composite snippet that has been segmented. 
         FIGS. 4B-4D  are pictorial diagrams illustrating an exemplary segmentation of the constituent images. 
         FIGS. 5A-5C  are pictorial diagrams illustrating exemplary constituent images. 
         FIG. 5D  is a pictorial diagram illustrating an exemplary composite image. 
         FIG. 5E  is a pictorial diagram illustrating exemplary peak masks derived from the composite image. 
         FIG. 5F  is a pictorial diagram illustrating identification of exemplary peaks using the peak masks previously formed. 
         FIG. 5G  is a pictorial diagram illustrating an exemplary snippet of the composite image identified by an exemplary peak mask. 
         FIGS. 5H-5J  are pictorial diagrams illustrating exemplary snippets of various individual constituent images overlaid with the exemplary peak mask. 
         FIGS. 5K-5M  are pictorial diagrams illustrating exemplary microaligned snippets of various constituent images. 
         FIGS. 5N-5O  are pictorial diagrams illustrating an exemplary microaligned, composite snippet. 
         FIG. 5P  is a pictorial diagram illustrating exemplary peak masks formed from the exemplary microaligned, composite snippet. 
         FIG. 5Q  is a pictorial diagram illustrating further exemplary snippets cut from the microaligned, composite snippet by using the exemplary peak masks. 
         FIG. 5R  is a pictorial diagram illustrating further segmentation of the exemplary snippets. 
         FIG. 5S  is a pictorial diagram illustrating exemplary peak masks. 
         FIGS. 5T-5V  are pictorial diagrams illustrating exemplary peaks on exemplary snippets of various individual constituent images. 
         FIGS. 6A-6C  are pictorial diagrams illustrating exemplary, constituent images. 
         FIGS. 6D-6F  are pictorial diagrams illustrating exemplary peak masks. 
         FIGS. 6G-6I  are pictorial diagrams illustrating identified relationships among the peak masks without the use of a composite image. 
         FIG. 6J  is a pictorial diagram illustrating a matrix of peak masks. 
         FIG. 6K  is a pictorial diagram illustrating selection of consensus peak masks in accordance with one embodiment of the present subject matter. 
         FIGS. 6L-6N  are pictorial diagrams illustrating peaks from various constituent images as detected by various peak masks. 
         FIGS. 6O-6Q  are pictorial diagrams illustrating peaks from various constituent images as detected by various consensus peak masks in accordance with one embodiment of the present subject matter. 
         FIGS. 7A-7N  are process diagrams illustrating a method for iteratively redefining regions of interest to discover biological features. 
         FIG. 8  is a block diagram illustrating a representative computing environment suitable for implementation of the disclosed methods. 
     
    
    
     DETAILED DESCRIPTION 
     As used in this application and in the claims, the singular forms “a,” “an,” and “the” include the plural forms unless the context clearly dictates otherwise. Additionally, the term “includes” means “comprises.” Further, the term “coupled” does not exclude the presence of intermediate elements between the coupled items. 
     The systems, apparatus, and methods described herein should not be construed as limiting in any way. Instead, the present disclosure is directed toward all novel and non-obvious features and aspects of the various disclosed embodiments, alone and in various combinations and sub-combinations with one another. The disclosed systems, methods, and apparatus are not limited to any specific aspect or feature or combinations thereof, nor do the disclosed systems, methods, and apparatus require that any one or more specific advantages be present or problems be solved. Any theories of operation are to facilitate explanation, but the disclosed systems, methods, and apparatus are not limited to such theories of operation. 
     Although the operations of some of the disclosed methods are described in a particular, sequential order for convenient presentation, it should be understood that this manner of description encompasses rearrangement, unless a particular ordering is required by specific language set forth below. For example, operations described sequentially may in some cases be rearranged or performed concurrently. Moreover, for the sake of simplicity, the attached figures may not show the various ways in which the disclosed systems, methods, and apparatus can be used in conjunction with other systems, methods, and apparatus. Additionally, the description sometimes uses terms like “produce” and “provide” to describe the disclosed methods. These terms are high-level abstractions of the actual operations that are performed. The actual operations that correspond to these terms will vary depending on the particular implementation and are readily discernible by one of ordinary skill in the art. 
     Mass spectrometry can be coupled with other separation techniques such as liquid chromatography, gel electrophoresis, gas chromatography, and ion diffusion. Data obtained by combinations of such techniques can be represented as n-dimensional data sets. For example, a chromatographic retention time and a mass-to-charge ratio of a mass spectrum can be used as coordinates of first and second axes. Signal intensities associated with these coordinates can be represented as a two-dimensional array of data values, and such data can be referred to as images or image data. 
     Mass spectrometry and liquid chromatography are two important separation techniques that can be applied to the evaluation of biological samples, and LC/MS data sets (referred to herein as images or image data) are used in the following description, but other types of data can be used. Typical biological specimens include mixtures of proteins, carbohydrates, lipids, and metabolites, but other chemical or biological specimens can be similarly investigated, and the disclosed methods and apparatus can be applied to non-biological specimens as well. 
     Some examples of the disclosed methods and apparatus pertain to pharmaceutical research. Pharmaceutical medical research is often directed to determining biological chemicals that are an indicator of a physiological state, such as a disease state or a response to treatment with a medication. A set of one or more of such chemicals is called a biomarker. Biomarkers may be used to diagnose disease or other physiological states. 
     Biomarkers can be used in a laboratory as substitutes for clinical endpoints, and such biomarkers are referred to as surrogate endpoints. Surrogate endpoints may be used to develop medicines without involving human or even animal subjects. Drug development may begin by testing for a surrogate endpoint in a test tube. 
     A common scenario is to process a biological specimen such as a biopsy specimen, blood, saliva, amniotic fluid, etc such that the complex chemical mixture associated with the specimen can be introduced into liquid chromatography/mass spectrometry instruments. The resulting data (signal intensity as a function of retention time and m/z ratio) is then used to identify the biochemicals in this complex mixture. The disclosed methods and apparatus permit identification of the more biologically relevant data by selecting only portions of the data. Specifically, the methods and apparatus permit identification of a signal of interest of one or more chemicals from the LC/MS data set, increased signal to noise ratio, typically by isolating or extracting the portion of the signal of interest from noise or other extraneous signals that can contaminate the signal of interest. Signal portions associated with one or more moieties can be removed or partially removed to permit investigation of additional signal features. For example, peaks associated with a particular moiety can be removed so that other peaks can be more clearly revealed. 
     Processing analytical data as disclosed provides superior isolation and identification and better quantification of chemicals in a complex mixture. A researcher can repeat this analysis process for healthy and diseased subjects and/or for untreated and treated subjects. Based on differences in chemicals constituents between a healthy and a diseased subject or between a treated and treated subject identified in this manner, a biomarker can be defined. In some cases, this biomarker can serve as a surrogate endpoint for use a substitute for a clinical endpoint. Such biomarkers may be used as diagnostic and treatment indicators in drug development. 
     Representative embodiments are described herein with respect to images based on liquid chromatography (LC) and mass spectrometry (MS). Typically, signal intensity is recorded as a function of LC retention time and MS mass-to-charge ratio (m/z), and this recorded signal intensity can be stored or processed as an image data array. For convenience herein, retention time is generally arranged along a horizontal (x-axis) and m/z is arranged along a vertical (y-axis). In other examples, other types of data are used instead of or in addition to LC retention time or m/z ratio. For example, gas chromatography (GC), ion spectroscopy, gel electrophoresis, ion diffusion, or mass spectroscopy time-of-flight data can be used. 
     In the following examples, analytical results associated with evaluation of biological samples are captured as digital images called replicates. Intensity variations are littered throughout the replicates. As noted above, conventional evaluation techniques are unable to identify corresponding relationships between various intensity variations among the replicates. While so-called “time warping” can be used to align these intensity variations to better identify those variations that are noise and reveal those intensity variations that are not noise, aligning some intensity variations results in the misalignment of other intensity variations, some of which might be important features. 
       FIG. 1A  illustrates a system  100  for processing biological samples so as to detect biological features. In a scientific experiment, each biological sample undergoes the same experiment (treatment condition) or different experiments (treatment conditions), which result in prepared biological samples  102 , and such prepared biological samples  102  are further processed by, for example, LC/MS equipment  104 , which applies various liquid chromatography and mass spectrometry processing. The results are one or more LC/MS images or constituent images that are further processed by an iterative regional processor  105 , which includes hardware and/or software executing on one or more computing devices. The processed regions provided by the iterative regional processor  105  are further processed by a region of interest processor  106 , which includes hardware and/or software executing on one or more computing devices. Various scores based on geometry and correlation allow the system  100  to classify biological features extracted from the prepared biological samples  102  to identify one or more biological features of interest  110 . 
       FIG. 1B  illustrates a portion  112  among many portions of the iterative regional processor  105  in greater detail. The portion  112  helps to refine peak detection in composite images, in one embodiment, and without the use of composite images, in another embodiment. The portion  112  identifies a region of interest, such as a peak, and uses information from multiple images to redefine a bounding area of a peak to encompass a suitably reproducible area of the peak across multiple images. Each image in the collection of images may be referenced as an LC/MS (liquid chromatography/mass spectrometry) image or a replicate or a constituent image or an LC/MS run. A peak is suitably expressed on one or more images. However, the bounding area may differ on each image to capture the peak. Each of these bounding areas may be different in size and location. Some of the bounding areas contain artifacts that may have been erroneously incorporated into the bounding area due to, for example, inadequate segmentation techniques. On some constituents, the bounding areas may be missing altogether. To facilitate better comparisons among boundary areas from different images, the bounding areas should suitably be made similar in size across different images. This localizes a pertinent region of interest to an exemplary boundary area that can be used to supplant different boundary areas for images. This exemplary boundary area represents a tool to illuminate desired peaks of various images. 
     The portion of the iterative regional processor  110  includes a reconciliator  114 . The reconciliator  114  identifies an eluted species over multiple constituent images. In other words, one or more constituent images may contain the eluted species of interest, and also many differently eluted species, often within very close proximity to the eluted species of interest. The difficulty is to associate one peak on one constituent image with other peaks on other constituent images, which represent the exactly same eluted species. The reconciliator  114  assists with this identification. One suitable implementation, as would be appreciated by one of ordinary skill in the art, is the use of peak reassembly techniques. Such techniques allow correlation of peaks from multiple images that typically cannot be superimposed by macroalignment. Other suitable techniques are possible. These constituent images can be used to produce a composite image, in one embodiment, by a suitable technique, such as averaging or maximal projection of the constituent images to form the composite image. Variations, such as shapes and intensities of spots on the composite image determine feature boundaries. Spots are intensity peaks that constitute a contiguous area in the constituent image. Multiplexed constituent images form a composite image. In such an image, peaks from constituent images may overlap or be in close proximity to peaks in other constituent images. Peaks that correspond to each other in this manner are presumed to result from the same chemical. Corresponding peaks from different constituent images overlap or are in close proximity on the composite image, due to variation in elution time, even after a possible macro-alignment. In another embodiment, analysis is made using the constituent images without the composite image. 
     The portion  112  includes a microaligner  116 . The microaligner  116  aligns in the retention time dimension to overcome an uncertainty or inadequate calibration associated with a liquid chromatography process. In one embodiment, the microaligner  116  aligns pixels of one region of interest with pixels from the same virtual region of interest on other constituent images, in the retention time dimension. Other parts of the constituent image and other regions of interest on the constituent image are not considered and are not affected even if these parts of regions of interest have the same retention time. This focus on a single region of interest at a time allows alignment that is not possible with conventional time warping. 
     The microaligner  116  aligns regions of interest in the constituent images whose boundaries are derived from a previously detected region of interest. The detection may have occurred using the composite image, in one embodiment, but in another embodiment, the composite image need not be used. Microalignment of regions of interest in the constituent images may aid in correlation of peaks and regional scoring, which reveals clues such as biological features or a lack thereof. Redefining the boundaries of regions of interest after microalignment, such as by the portion  112 , may help reduce, remove or minimize noise in the region of interest by tightening the region of interest, thereby excluding noise that was part of the original region of interest. 
     The microaligner  116  may refine alignment of regions of interest even in the absence of a composite image. In this instance, regions of interest on various constituent images are correlated and presumed to have originated from the same chemical species if their macroaligned bounded areas overlap or are in close proximity to each other. Such regions of interest correspond to each other on these constituent images. The microaligner  116  then aligns a set of corresponding regions of interest. One use of a composite image in microalignment is to associate regions of interest from multiple constituent images. In this use, the composite image identifies a common region of interest, which can be applied to all constituents considering their differences in retention time. If sets of corresponding regions of interest can be identified without a composite image, then those sets of corresponding regions of interest can be microaligned. 
     The iterative regional processor  106  includes a regional redefiner  118 . Because of varying boundaries to capture regions of interest in various constituent images, these regions of interest may include artifacts that are not of interest. Such artifacts are noise, such as surrounding background and neighboring contaminates, to be contrasted with a signal that includes pixels of interest. By creating a composite snippet, the original region of interest can be redefined such that it is more likely to include more of pixels of interest and fewer pixels not of interest, such as noise. With less noise, the signal becomes better identified and enables better scoring to reveal biological clues leading to scientific discovery. The regional redefiner  118  alters bounding areas for peaks across constituent images, all pointing to the same clue to a biological feature of interest. The result is a common bounding area which helps to facilitate quantitation of peaks by improving statistical models for evaluating and comparing peaks. 
       FIG. 2D  illustrates a composite image formed from constituent images illustrated by  FIGS. 2A-2C . There are various suitable techniques for combining constituent images to form the composite image, such as averaging or maximal projection. Suitably, an averaging is used to enhance the signal-to-noise ratio. A segmentation process is applied to provide granular areas that are likely to contain peaks. Peaks shown in the figures are represented as series of nested ellipses. Inner ellipses indicate increased signal intensity and thus the elliptical representation is similar to topographic map. An intensity maximum is situated within the smallest ellipse. Ellipse spacing corresponds to constant signal intensity steps. Next, a suitable association process, such as peak reassembly, is executed to yield entities localized by bounding areas that are likely to reflect individual species. These bounding areas are represented as rectangular boxes in  FIG. 2D , but bounding areas can be elliptical, circular, square, oval, polygonal, or other regular or irregular contiguous shapes. Referring to  FIGS. 2A-2C , the bounding areas can be projected onto the constituent images and the information that is bounded by corresponding bounding areas is considered reconciled in the sense that a peak that is bounded by one bounding area in one constituent image is considered to be associated with another peak that is bounded by the same bounding area in another constituent image. 
       FIG. 3A  illustrates snippets of the constituent images found in  FIGS. 2A-2C . As can be appreciated, these snippets are not microaligned. The peaks in these figures are visually represented as series of ellipses as described above. The peaks in the upper snippet are flushed to the right. The peaks in the middle snippet are flushed to the left, and the peaks in the lower snippet tend toward the left. To begin the microalignment process, a bounding area of the same size is applied to these snippets. See  FIG. 3A . Next, a master snippet is chosen (in this example, the upper snippet) to which other snippets (the middle snippet and the lower snippet) will be microaligned.  FIG. 3B  illustrates that the middle snippet and the lower snippet have been shifted to the left. Two arrows with varying lengths indicate the extent to which shifting has occurred to achieve microalignment with the master snippet (the upper snippet).  FIG. 3C  illustrates a composite snippet formed from the microaligned snippets illustrated by  FIG. 3B . 
       FIG. 4A  illustrates the composite snippet formed from the microaligned snippets, which has undergone image segmentation to identify two peaks that are adjacent to each other. The segmentation process reveals adjacent bounding areas that redefine a region of interest. The bounding areas are represented as rectangular boxes, but other shapes can be used.  FIGS. 4B-4D  illustrate the application of the bounding areas identified from the microaligned, composited, segmented image to the constituent images. This application redefines the boundaries of the peaks of interest. 
       FIGS. 5A-5C  illustrate peaks across constituent images. These peaks are not associated with each other. Thus, no conclusion can be made as to whether some of them point to the same clue to illuminate biological features of interest. The peaks are visually represented as ellipses that contain other ellipses. Each inner ellipse visually illustrates an increased intensity like a topographic map so that the smallest ellipse contained by other larger ellipses contains an apex of intensity maximum. It can be assumed that the constituent images of  FIGS. 5A-5C  have been macroaligned, which macroalignment appears to be imperfect because residual localized misalignment remains. 
       FIG. 5D  illustrates a composite image formed from the multiplexing of constituent images of  FIGS. 5A-5C .  FIG. 5E  illustrates two spots formed from executing a segmentation process on the composite image of  FIG. 5D . The two spots (upper mask and lower mask) have bounding areas that identify pieces of information to be further processed by regional redefinition in accordance with various embodiments of the present subject matter. See  FIG. 5F .  FIG. 5G  illustrates a selection of the lower mask, which identifies pieces of information in the composite image that is elongated and is referred to as Spot  1  on  FIG. 5F . Using the bounding area of the mask, additional pieces of information are identified by the application of the bounding area of the mask on the constituent images to form snippets. See  FIGS. 5H-5J . 
     The snippets are then microaligned. See  FIGS. 5K-5M  where the snippets are uneven in their position so as to microalign peaks contained by the snippets.  FIG. 5N  illustrates a composite snippet formed from the microaligned snippets of  FIGS. 5K-5M .  FIG. 5O  is a representation of the composite snippet to ease the discussion of the following figures.  FIG. 5P  illustrates a segmentation of the composite snippet to form two masks.  FIG. 5Q  illustrates the use of the bounding areas of the two masks to differentiate the two regions of interest of the composite snippet. The process above is repeated so that  FIG. 5R  illustrates the identification of three distinct peaks.  FIG. 5S  illustrates masks formed from these distinct peaks. The masks are applied to the original constituent images to reveal peaks that are associated with each other, hence illuminating clues to biological features of interest. The masks of  FIG. 5S  are applied to the three lower peaks in each of  FIGS. 5T-5V . The mask for the upper spot is derived from Spot  2  on  FIG. 5F .  FIGS. 5T-5V  have regionally redefined peaks that correspond among the constituents. Corresponding peaks among  FIGS. 5T-5V  are  502 A- 502 C,  504 A- 504 C,  506 A- 506 C, and  508 A- 508 C. 
       FIGS. 6A-6C  illustrate peaks across constituent images. These peaks are not associated with each other. Thus, no conclusion can be made whether some of them point to the same clue to illuminate biological features of interest. The peaks are represented as ellipses that contain other ellipses. Each inner ellipse visually illustrates an increased intensity like topographic maps as described above.  FIGS. 6D-6F  are masks of spots resulted from a segmentation process, which is executed on the constituent images of  FIGS. 6A-6C .  FIGS. 6G-6I  illustrate the spot masks but those masks that have a relationship with each other are visually tied by a line that crosses among constituent images. 
       FIG. 6J  illustrates a matrix comprising columns and rows. The intersection of each column and each row stores a spot mask. Each column points to spot masks that are related to each other, probably revealing peaks that point to the same biological clue or species. Each row points to spot masks that are on the same constituent image. For example, the first row points to spot masks that come from the first constituent image ( FIG. 6A ); the second row points to spot masks that come from the second constituent image ( FIG. 6B ); and the third row points to spot masks that come from the third constituent image ( FIG. 6C ).  FIG. 6K  illustrates another matrix that has multiple columns and one row. The one row represents consensus spot masks that are associated with peaks corresponding to different biological clues or chemical species. In one embodiment, the consensus spot masks are the largest of all the spot masks in the columns of the matrix of  FIG. 6J . In another embodiment, a union of the spot masks of each column of the matrix of  FIG. 6J  is the consensus spot mask for that column. In a third embodiment, the consensus spot mask is the largest ellipse that can be fitted into the union of spot masks of the same column of the matrix of  FIG. 6J . Related peaks are the peaks  602 A- 602 C,  604 A- 604 C,  606 A- 606 C, and  608 A- 608 C, respectively, as shown in the constituent images of  FIGS. 6L-6N .  FIGS. 6O-6Q  illustrate constituent images with peaks that are related, identified, and bounded in accordance with the consensus spot masks of the matrix of  FIG. 6K . 
     The converged peak masks are placed cookie-cutter fashion onto the constituent images. The location of the placement takes into consideration macroalignment and microalignment. In the example of  FIG. 5 ,  FIGS. 5T-5V  represent the regionally redefined peak detections that are the result of this process. In the example of  FIG. 6 ,  FIGS. 6L-6Q  represent the regionally redefined peak detections that are the result of the process in which the intermediate microaligned composite image was not generated. 
       FIGS. 7A-7N  illustrate a method  7000  for iteratively redefining regions of interest to discover biological features. From a start block, the method  7000  proceeds to a set of method steps  7002 , defined between a continuation terminal (“terminal A”) and an exit terminal (“terminal B”). The set of method steps  7002  describes the execution of a set of reconciliation steps from which data is derived from prepared biological samples. The reconciliation step associates peaks from different constituents. See  FIGS. 7B-7D . Images of such constituents are exemplified in  FIGS. 5A-5C . It can be assumed that these constituent images have been macroaligned, which macroalignment is imperfect due to residual local misalignment. Regarding the reconciliation process for a composite image as shown in  FIG. 5D , image segmentation performed on a composite image produces a set of peaks as shown in  FIG. 5E . In a desired result, a single chemical species is represented by one peak and each peak represents only one chemical species. However, over-segmentation or peak merging may occur. Over-segmentation may lead to multiple peaks that may in fact represent the same single species. Such over-segmented peaks need to be reassembled back into a single peak. A peak reassembler executing a peak reassembly process may suitably be used to reassemble an over-segmented peak. Conversely, mis-alignments among the constituents, as exemplified in  FIGS. 5A-5C  may cause merging of peaks on a composite image that point to different chemical species. This situation is exemplified in  FIGS. 5E-5F , where a conglomeration of peaks, referred to as Spot  1  in  FIG. 5F  results from multiplexing  FIGS. 5A-5C . In these cases, the separation of species is achieved through microalignment followed by a segmentation. Regarding the reconciliation process for a set of constituent images without the composite image, image segmentation is executed on each individual constituent image alone to create a list of peaks for each respective constituent image. The method then associates a peak on a constituent image with other peaks on other constituent images if they point to the same species. 
     From terminal A ( FIG. 7B ), the method  7000  proceeds to block  7008  to execute LC/MS processing to produce a set of LC/MS images (constituent images). At block  7010 , the method optionally executes a macroalignment process, such as a time-warp algorithm, to align the set of constituent images. Such constituent images are pictorially exemplified in  FIGS. 5A-5C . It can be assumed that these constituent images have been macroaligned, which macroalignment appears to be imperfect because residual localized misalignment remains. The method then proceeds to decision block  7012  where a test is performed to determine whether the method is using only constituent images as input for regional redefinition. If the answer to the test is No, the method proceeds to another continuation terminal (“terminal A 1 ”). (In other words, the method selects to use a composite image instead of using its constituent images to determine peak masks.) Otherwise (in which case the method selects to use only constituent images instead of a composite image based on the constituent images), if the answer to the test is Yes, the method proceeds to block  7014  where the method  7000  identifies regions of interest in the constituent images by using peak detection to form a list of peaks for each constituent image. At block  7016 , the method considers all peaks, such that a peak P 1  on a constituent image R 1  is associated with other peaks, identifying the same species P on the remaining constituent images R 2 , . . . , R N . The method then enters exit terminal B. 
     From terminal A 1  ( FIG. 7C ), a composite image is formed from the set of constituent images. See block  7018 . Such a composite image is pictorially exemplified in  FIG. 5D . The method  7000  then identifies regions of interest in this composite image by detecting variations in the composite image (such as peak detection). See block  7020 . Peak detection results in peak masks that define the location and boundaries of peaks.  FIG. 5E  is a pictorial representation of example peak masks that could be derived from the composite image exemplified in  FIG. 5D . At block  7022 , the method creates a collection of peak masks. Initially, all peaks are considered to be not converged. Next, the method proceeds to another continuation terminal (“terminal A 2 ”). From terminal A 2 , the method proceeds to decision block  7024  where a test is performed to determine whether all peak masks have converged. If the answer to the test at decision block  7024  is Yes, the method proceeds to another continuation terminal (“terminal E 3 ”) and terminates execution. Otherwise, if the answer to the test at decision block  7024  is No, and the method proceeds to block  7026  where the method selects a non-converged peak mask from the collection of peak masks. The method then enters another continuation terminal (“terminal A 3 ”). 
     From terminal A 3  ( FIG. 7D ), the method identifies the region of interest in the composite image that contains the peak mask selected in block  7026 . This region of interest is termed the original region of interest. See block  7028 .  FIG. 5G  exemplifies in pictorial form such an original region of interest. At block  7030 , the method identifies regions of interest in the constituent images whose boundaries and locations relate to the original region of interest in the composite image. The region of interest on each constituent image is determined by the composite image and then projected in a cookie-cutter fashion onto the constituent images considering their respective macroalignments.  FIGS. 5H-5J  exemplify the respective regions of interest for each constituent that were identified using the exemplified original region of interest from  FIG. 5O . The method measures the width of the original region of interest (in the retention time dimension) in the composite image. See block  7032 . The method further expands the width of each identified region of interest backward and forward in the retention time dimension by the measured width resulting in a threefold increase. See block  7036 . At block  7038 , the method optionally reduces the height of each identified region of interest to focus on a chromatogram at a central mass/charge modified by a tolerance. In summary, the above steps describe the use of the non-converged composite peak mask to identify an image snippet within each constituent image, resulting in a collection of image snippets. 
     From terminal B ( FIG. 7A ), the method  7000  proceeds to a set of method steps  7004 , defined between a continuation terminal (“terminal C”) and an exit terminal (“terminal D”). The set of method steps  7004  executes a microalignment process that aligns regions of interest. From terminal C ( FIG. 7E ), the method  7000  proceeds to decision block  7042  where a test is performed to determine whether the master snippet is determined from among the constituents by alignment to the composite image. If the answer to the test at decision block  7042  is NO, the method  7000  proceeds to block  7044  where a suitable master snippet is selected from among the constituent and composite snippets by other means including a random choice. The method then continues to another continuation terminal (“terminal C 7 ”). If, instead, the answer is YES to the test at decision block  7042 , the method continues to another continuation terminal (“terminal C 2 ”). 
     From terminal C 2  ( FIG. 7E ), the method  7000  proceeds to block  7046  where the method begins to find a region of interest (as the perspective master snippet) in the subset of constituent images that correlates with the original region of interest. At block  7048 , the original region of interest is a matrix U=u ij  and the prospective master snippet is defined as V=v ik . The variable k has a range from j−n, . . . , j+n, where n is the width of the original region of interest (in pixels). See block  7048 . The variable i ranges from 1 to m in the mass/charge dimension. See block  7050 . The variable j ranges from 1 to n along the retention time dimension. See block  7050 . At block  7052 , v(q) is a vector derived from the prospective master snippet. The variable q represents the retention time shift and it ranges from −n, . . . , n. See block  7052 . The method  7000  then continues to another continuation terminal (“terminal C 3 ”). 
     From terminal C 3  ( FIG. 7F ), the method prepares to calculate normalized cross-correlation maximum t 1 (q). See block  7054 . At block  7046 , the method calculates the term 
     
       
         
           
             
               u 
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             = 
             
               
                 1 
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                 ⁢ 
                 
                   
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                       u 
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                     . 
                   
                 
               
             
           
         
       
     
     The term ū is the result of the above mathematic operation. At block  7058 , the method further calculates the following term 
     
       
         
           
             
               
                 v 
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     The resulting  v(q)  is the result of such a mathematical operation. The method  7000  proceeds to block  7060  where the method calculates t 1 (q) from the following expression: 
     
       
         
           
             
               
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     The method  7000  continues to another continuation terminal (“terminal C 4 ”). From terminal C 4  ( FIG. 7F ), the method proceeds to block  7062  where the method prepares to calculate linear correlation coefficient maximum t 2 (q), which is a quotient of a dividend and a divisor. At block  7064 , the dividend is calculated as follows: 
     
       
         
           
             
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     At block  7066 , the divisor is calculated as follows: 
     
       
         
           
             
               
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     The method  7000  then continues to another continuation terminal (“terminal C 5 ”). From terminal C 5  ( FIG. 7G ), the method prepares to calculate normalized least-square difference minimum t 3 (q). See block  7068 . The method calculates the following expression: 
     
       
         
           
             
               
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     See block  7070 . The method then continues to another continuation terminal (“terminal C 6 ”). From terminal C 6  ( FIG. 7G ), the method proceeds to block  7072  where a score for the region of interest is selected among normalized cross-correlation maximum, linear correlation coefficient maximum, and normalized least-square difference minimum. The method  7000  proceeds to decision block  7074  wherein a test is performed to determine whether all constituents were evaluated for this region of interest. If the answer to the test at decision block  7074  is NO, the method proceeds to terminal C 2  and loops back to block  7046  where the above-identified processing steps are repeated. Otherwise, if the answer to the test at decision block  7074  is YES, then the method proceeds to block  7076  where the region of interest with a score that best correlates with the original region of interest in a composite image is selected as the master snippet for micro-alignment. The method then continues to another continuation terminal (“terminal C 7 ”). 
     From terminal C 7  ( FIG. 7H ), the method selects a region of interest (an image snippet) in a constituent image in the subset for micro-alignment with the master snippet. See block  7078 . The master snippet is a matrix U=u ij  and the image snippet to be micro-aligned is defined as V=v ik . See block  7080 . Furthermore, n is the width of the original region of interest (in pixels) and i ranges from 1 to m in the mass/charge dimension, and j ranges from 1 to n along the retention time dimension, and k ranges from j−n to j+n along the retention time dimension. See blocks  7082 ,  7084 . v(q) is derived from the image snippet v ik  by shifting k (in the retention time dimension) by q, which ranges −m, . . . , n. See block  7086 . q is calculated based on a suitable correlation of the intensity of v(q) with the master snippet u ij  excluding any pixels that do not match up because k−q≠j. See block  7088 . Various strategies to time alignment may be used. What follows are some example strategies. One value of q is calculated such that t 1 (q) is maximized using the normalized cross-correlation maximum described between terminals C 3 , C 4 . See block  7090 . The method  7000  proceeds to another continuation terminal (“terminal C 11 ”). 
     From terminal C 11  ( FIG. 7I ), another value of q is calculated such that t 2 (q) is maximized using the linear correlation coefficient maximum described between terminals C 4 , C 5 . See block  7092 . A further value of q is calculated such that t 3 (q) is minimized using the normalized least-square difference minimum described between terminals C 5 , C 6 . See block  7094 . The method  7000  then proceeds to another continuation terminal (“terminal C 8 ”). From terminal C 8  ( FIG. 7J ), the method proceeds to decision block  7096  where a test is performed to determine whether there is a correlation that identifies a suitable microalignment. If the answer to the test at decision block  7096  is YES, the shifted image snippet that provides a suitable correlation with the master snippet is kept for further processing. See block  7098 . The method then continues to another continuation terminal (“terminal C 9 ”). If the answer to the test at decision block  7096  is NO, the method continues to another decision block  7100  where a test is performed to determine whether centroid alignment should be used. If the answer to the test at decision block  7100  is YES, the method shifts the retention time of the image snippet such that its maximum or centroid coincides with the maximum or centroid of the master snippet. See block  7102 . The method then continues to terminal C 9 . If the answer to the test at decision block  7100  is NO, the method continues to another continuation terminal (“terminal C 10 ”). 
     From terminal C 10  ( FIG. 7K ), the method proceeds to decision block  7104  where a test is performed to determine whether another method of alignment should be used, such as adaptive alignment. If the answer to the test at decision block  7104  is YES, the method executes the other form of alignment to micro-align the image snippet with the master snippet. See block  7106 . The method then continues to terminal C 9 . If the answer to the test at decision block  7104  is NO, the method continues to terminal C 9  and further proceeds to decision block  7108  where a test is performed to determine whether there is another region of interest for micro-alignment. If the answer to the test at decision block  7108  is YES, then the method proceeds to terminal C 7  and loops back to block  7082  where the above-identified processing steps are repeated. Otherwise, if the answer to the test at decision block  7108  is NO, then the method continues to exit terminal D.  FIGS. 5K-5M  are pictorial examples of the microaligned regions of interest given the image snippets exemplified in  FIGS. 5H-5J . 
     From terminal D ( FIG. 7A ), the method  7000  proceeds to a set of method steps  7006  defined between a continuation terminal (“terminal E”) and an exit terminal (“terminal F”). The set of method steps  7006  executes a regional redefinition process to focus on a boundary area that captures a better signal-to-noise ratio and is suitable for a subsequent scoring process and scientific evaluation. From terminal E ( FIG. 7L ), the method proceeds to decision block  7110  where a test is performed to determine whether the method is using a microaligned composite image as input. If the answer to the test at decision block  7110  is NO, the method proceeds to a continuation terminal (“terminal E 2 ”). Otherwise, if the answer to the test at decision block  7110  is YES, then the method executes steps to create a composite snippet (different from the original region of interest in the composite image). See block  7112 .  FIGS. 5K-5M  are pictorial examples of the microaligned regions of interest that resulted from the microaligned process of method steps  7004 .  FIG. 5N  is a pictorial example of the composite snippet derived from these microaligned regions of interest. 
     At block  7114 , the composite snippet comprises a two-dimensional representation of each peak P from various constituent images R 1 , . . . , R N . The method optionally executes a smoothing step to remove anomalous noise. See block  7116 . At block  7118 , the method executes variation detection (peak detection), such as thresholding, watershed, horizontal and/or radial edge detection by a Laplace of Gaussian convolution, to identify new peak masks in the composite snippets. A peak reassembly step is executed to stitch together peaks that may have been incorrectly segmented into multiple portions. See block  7120 . The method then enters into another continuation terminal (“terminal E 1 ”). 
     From terminal E 1  ( FIG. 7M ), the method creates a new peak mask from the microaligned composite snippet. See block  7122 .  FIG. 5P  exemplifies the new peak masks that were derived from  FIG. 5O  by peak detection. The method proceeds to decision block  7124  where a test is performed to determine whether the new peak mask is identical to the non-converged peak mask that was selected in block  7026 . If the answer to the test at decision block  7124  is NO, the method removes the selected non-converged peak mask. See block  7126 . The method then proceeds to block  7128  where the method adds the new peak masks from the micro-aligned composite snippet to the collection of non-converged peak masks. In the example of  FIG. 5 , the two peak masks, shown both in  FIG. 5P  and  FIG. 5Q , differ from the initial micro-aligned composite peak mask, that is the lower spot in  FIG. 5E . In this example, the peak mask that represents the lower spot in  FIG. 5E  would be removed from the non-converged collection of peak masks, but would not be marked as converged. The two new peak masks, shown both in  FIG. 5P  and  FIG. 5Q , would be added to the non-converged collection. The method then proceeds to terminal A 2  and skips back to decision block  7024  where the above identified processing steps are repeated. Otherwise, if the answer to the test at decision block  7124  is YES, the method proceeds to block  7130  where the method marks the peak mask as a converged peak mask. The method then proceeds to terminal A 2  and skips back to decision block  7024  where the above identified processing steps are repeated. Given the pictorial example of  FIG. 5 , the two peak masks shown in  FIG. 5P  will be resolved into the three peak masks, shown both in  FIG. 5R  and  FIG. 5S , by the same processes that produced the two peak masks shown in  FIG. 5P  from  FIG. 5E . 
     From terminal E 2  ( FIG. 7N ), the method proceeds to block  7132 . For each peak P representing a species, the method modifies an initial peak boundary of peak P on a constituent image R to a consensus peak boundary. At block  7134 , the method assigns the larger bound of the consensus peak boundary as the union of the bounding region of peak P across all constituent images. At block  7136 , the method forms a common region of interest, hence a regional redefinition. This process is shown in pictorial form in  FIGS. 6J-6K . The method proceeds through terminal E 3  to block  7138 , where the regionally redefined peak masks are applied to the constituent images using their respective alignments.  FIGS. 5T-5V  and  FIGS. 6L-6Q  depict the results of the iterative processing for the example in  FIG. 5  and  FIG. 6 , respectively. The method then continues to terminal F and terminates execution. 
       FIG. 8  and the following discussion are intended to provide a brief, general description of an exemplary computing environment in which the disclosed technology may be implemented. Although not required, the disclosed technology is described in the general context of computer-executable instructions, such as program modules, being executed by a personal computer (PC). Generally, program modules include routines, programs, objects, components, data structures, etc., that perform particular tasks or implement particular abstract data types. Moreover, the disclosed technology may be implemented with other computer system configurations, including hand-held devices, multiprocessor systems, microprocessor-based or programmable consumer electronics, network PCs, minicomputers, mainframe computers, and the like. The disclosed technology may also be practiced in distributed computing environments where tasks are performed by remote processing devices that are linked through a communications network. In a distributed computing environment, program modules may be located in both local and remote memory storage devices. With reference to  FIG. 8 , an exemplary system for implementing the disclosed technology includes a general purpose computing device in the form of an exemplary conventional PC  800 , including one or more processing units  802 , a system memory  804 , and a system bus  806  that couples various system components including the system memory  804  to the one or more processing units  802 . The system bus  806  may be any of several types of bus structures including a memory bus or memory controller, a peripheral bus, and a local bus using any of a variety of bus architectures. The exemplary system memory  804  includes read only memory (ROM)  808  and random access memory (RAM)  810 . A basic input/output system (BIOS)  812 , containing the basic routines that help with the transfer of information between elements within the PC  800 , is stored in ROM  808 . 
     The exemplary PC  800  further includes one or more storage devices  830  such as a hard disk drive for reading from and writing to a hard disk, a magnetic disk drive for reading from or writing to a removable magnetic disk, and an optical disk drive for reading from or writing to a removable optical disk (such as a CD-ROM or other optical media). Such storage devices can be connected to the system bus  806  by a hard disk drive interface, a magnetic disk drive interface, and an optical drive interface, respectively. The drives and their associated computer-readable media provide nonvolatile storage of computer-readable instructions, data structures, program modules, and other data for the PC  800 . Other types of computer-readable media which can store data that is accessible by a PC, such as magnetic cassettes, flash memory cards, digital video disks, CDs, DVDs, RAMs, ROMs, and the like, may also be used in the exemplary operating environment. 
     A number of program modules may be stored in the storage devices  830  including an operating system, one or more application programs, other program modules, and program data. A user may enter commands and information into the PC  800  through one or more input devices  840  such as a keyboard and a pointing device such as a mouse. Other input devices may include a digital camera, microphone, joystick, game pad, satellite dish, scanner, or the like. These and other input devices are often connected to the one or more processing units  802  through a serial port interface that is coupled to the system bus  806 , but may be connected by other interfaces such as a parallel port, game port, or universal serial bus (USB). A monitor  846  or other type of display device is also connected to the system bus  806  via an interface, such as a video adapter. Other peripheral output devices, such as speakers and printers (not shown), may be included. 
     The PC  800  may operate in a networked environment using logical connections to one or more remote computers, such as a remote computer  860 . In some examples, one or more network or communication connections  850  are included. The remote computer  860  may be another PC, a server, a router, a network PC, or a peer device or other common network node, and typically includes many or all of the elements described above relative to the PC  800 , although only a memory storage device  862  has been illustrated in  FIG. 8 . The personal computer  800  and/or the remote computer  860  can be connected to a logical a local area network (LAN) and a wide area network (WAN). Such networking environments are commonplace in offices, enterprise-wide computer networks, intranets, and the Internet. 
     When used in a LAN networking environment, the PC  800  is connected to the LAN through a network interface. When used in a WAN networking environment, the PC  800  typically includes a modem or other means for establishing communications over the WAN, such as the Internet. In a networked environment, program modules depicted relative to the personal computer  800 , or portions thereof, may be stored in the remote memory storage device or other locations on the LAN or WAN. The network connections shown are exemplary, and other means of establishing a communications link between the computers may be used. Having described and illustrated the principles of our invention with reference to the illustrated embodiments, it will be recognized that the illustrated embodiments can be modified in arrangement and detail without departing from such principles. For instance, elements of the illustrated embodiment shown in software may be implemented in hardware and vice-versa. Also, the technologies from any example can be combined with the technologies described in any one or more of the other examples. In view of the many possible embodiments to which the principles of the invention may be applied, it should be recognized that the illustrated embodiments are examples of the invention and should not be taken as a limitation on the scope of the invention. For instance, various components of systems and tools described herein may be combined in function and use. We therefore claim as our invention all subject matter that comes within the scope and spirit of these claims. Alternatives specifically addressed in these sections are merely exemplary and do not constitute all possible alternatives to the embodiments described herein. While illustrative embodiments have been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention. 
     In view of the many possible embodiments to which the principles of the disclosed invention may be applied, it should be recognized that the illustrated embodiments are only preferred examples of the invention and should not be taken as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.