Overview

The MSI Analyte Browser provides capability to annotate, visualize and explore 2D mass spectrometry imaging data. The app takes analyte .txt files from the MaldiChrom utility in HDI as input. Additionally,the app can read .rte files output from the 2D real-time viewer plugin to HDI, or a generic CSV format with columns representing (scan_number, x_pixel_location, y_pixel_location, mz_0, mz_1, ..., mz_N) for N m/z bins.

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Changelog

• Version 2.1.1
  • Fixes bug that caused error loading certain types of analyte text files
• Version 2.1.0
  • Adds support for analyte files with IMS data
  • Reads TIC data from analyte files if present (HDI 1.7)
  • Mass annotation now accounts for the mass of an electron
  • Updated spectrum visualization figure
• Version 2.0.1
  • Fixes bug that caused ion image viewer to crash for square images
• Version 2.0.0
  • Major UI updates to group individual visualizations in tabs
  • Adds new RGB visualization feature
  • Adds new interactive/clickable pixel selection feature
  • Updated annotation matching algorithm for more intuitive results
  • Fixes visualizations for data with nonsquare pixel dimensions
  • Fixes bug in the "difference" spectrum" viewer that clipped missing peaks to -100%
  • Fixes bug that prevented the input of analyte data created in HDI with a target list
• Version 1.0.0
  • Initial release

Installation

MSI Analyte Browser is bundled with a Windows 10 installer. After downloading, be sure to uninstall any previous versions, then launch the installer by launching "Waters.MSIAnalyteBrowser.BundleInstaller.exe" [Note: do not launch "Waters.MSIAnalyteBrowser.Deployment.msi"]. After installation, the software will be installed as "Waters MSI Analyte Browser". This launches a window where the app service can be launched by pressing "Start". The app itself can then be accessed in a browser window at the local URL printed in the "Streamlit output" window. It should open a browser window to this address automatically, but in case it does not, you can manually copy and paste the address. To close, click "Stop" to stop the service, then close the app window.

Loading data

After the file-type extension is selected (.txt, .csv, or .rte), data are loading using the "Load data" form in the sidebar, using the "Browse files" button for local data, or selecting a sample file using the "Select example data" dropdown list. An uploaded local file will override a selected example file if both are selected. Once the data are loaded, metadata for the image dimensions, number of scans, pixel sizes, m/z range, and number of peaks are displayed below the "Load data" window in the sidebar. To speed up data uploading, the app will accept zip-compressed files provided there is only a single dataset within the zipped folder.

M/Z Annotation

MSI Analyte Browser provides tentative annotation of ion images based on heuristics to identify potential isotope images and matching to entries to the Human Metabolome Database(HMDB). These annotations are subject to the following user controls:

• Mass Accuracy (ppm)
  • Sets the search window for isotope detection and analyte look-up
• Isotope image R^2 threshold
  • Candidate isotope images must have a correlation to the monisotopic image greater than this threshold
• Ion mode
  • Negative or positive

Results are displayed in three tabs in the "M/Z Annotation" processing page:

1. The "Analyte annotation lookup" tab
   • This utility allows the user to choose a measured m/z value and view potential annotations across all potential adducts, searching by measured m/z, formula, or name.
2. The "Isotope results" tab
   • This table shows the list of measured m/z images identified as isotopes, together with the associated [M+0] image, N (the rounded mass difference to [M+0] in Da), and the R^2 value between the image intensities.
3. The "Closest match for each m/z" tab
   • This table contains a summary of annotation results for all measured m/z values in the dataset. It shows the number of matches and the formula and name of the closest match for all measured m/z values in the dataset, including isotope images.

Data Viewer

On the "Data viewer" processing page, visualizations are generated in two tabs:

1. The "Primary viewer" tab
   • This displays a 2D image of a specific m/z analyte selected in the "Image controls" panel. It also shows dotted-line crosshairs highlighting the currently selected pixel for the "Spectrum" tab. It allows for interactive exploration with various zoom and pan tools, as well as a hover tool which displays the (x,y) location and intensity when the mouse hovers over a pixel.
   • Clicking on a pixel will update the crosshairs and data for the Pixel Spectrum figure, which displays the analyte spectrum at a given (x,y) pixel location. It allows for interactive exploration with various zoom and pan tools, as well as a hover tool which displays the m/z value intensity, name and chemical formula with a guiding vertical line as the mouse moves across the m/z axis.
   • This window also contains controls to display either the "raw" or "difference" spectrum. "Raw" shows the raw spectrum counts at the specified pixel location, while "difference" shows the percent difference of the counts at the specified pixel and the average spectrum across all pixels.
2. The "Average spectrum" tab
   • This displays the analyte spectrum averaged across all pixel locations. It is implemented as a Bokeh figure, which allows interactive exploration with various zoom and pan tools, as well as a hover tool which displays the m/z value intensity, name and chemical formula with a guiding vertical line as the mouse moves across the m/z axis.

Additional controls for the Primary Viewer are located in the "Image controls" panel:

• "Select analyte"
  • This is a selection box that determines the m/z value visualized in the "Ion image" window. A specific value can be selected on through scrolling or typed directly into the input.
• "Select colormap"
  • This allows the user to change the colormap for the "Ion image". The "Name" dropdown menu allows the user to select one of six named matplotlib colormaps, and the "Display range" double slider allows for custom scaling. The default display values cover the entire dynamic range of the image.

Additional visualizations

Additional custom data visualizations are presented on this page, including:

1. The "Top analytes" tab
   • This displays a grid of the top 50 analytes, intended to guide selection of m/z values for further inspection in the "Ion image" tab. The top analytes are chosen by ranking m/z values by the ratio of the sum of their intensities inside and outside a custom object mask. Once chosen, the top images are ordered by m/z value and plotted as a grid in a single matplotlib figure.
2. The "RGB Viewer" tab
   • This allows for the creation of a single RGB color figure where each channel is assigned the intensities from a specific m/z ion image.
3. The "Ion image video" tab
   • This generates and displays an mp4 video fly-through video of every ion image in order of ascending m/z. The video is saved and cached after creation for quick reloading. Note that generating the video generally takes several minutes.

Visualization - RTE

If RTE data are uploaded, the visualizations for all XiCs are displayed in the main window of the "Data viewer" page. Each image displays the distribution of a specific m/z analyte and have the standard display controls in the "Image controls" panel. No additional visualizations are present for .rte data.