Manual
- 1 Opening MIMS images
- 2 Graphical Interface
- 3 ROI Manager
- 4 Menu Items - 4.1 File - 4.2 Edit - 4.3 View - 4.4 Utilities - 4.5 Corrections
- 5 Appendices
- 6 References
After launching ImageJ (or Fiji), place the mouse over the “Plugins” menu option and select the “Open MIMS Image” option. This action will open the OpenMIMS graphical user interface. You then open an image by clicking “File > Open MIMS Image” and navigate to the desired MIMS image file (files with a .im, .nrrd extension). Files can also be opened by dragging and dropping the file from a file browser. Only .nrrd files generated by the OpenMIMS plugin are guaranteed to be readable by the plugin. After the MIMS image is opened, each mass will appear in a separate window (example below) as well as the NRIMS Analysis Module window.
Two different mass images of the same field of view.
The NRIMS Analysis Module window contains 7 tabs:
- MIMS Data: Displays metadata and other information for the current file.
- Process: Used to generate ratio images and HSI maps.
- Contrast: Controls the contrast and brightness settings for mass, ratio and sum images.
- Stack Editing: Used for manipulating the mass images (applying x and y translations, dropping planes, etc).
- Tomography: Basic plotting of statistics for whole images or ROIs.
- Segmentation: Perform an algorithmic segmentation of an image.
- MIMS Log: Displays more header metadata and a record of the user’s actions and debug output.
A screenshot of the OpenMIMS application.
The MIMS Data tab displays a subset of the image’s meta-data including the absolute path of the current image, the number of masses and their AMU values, number of planes in the stack, position, date of image, user name, dwell time, duration, raster size and image size. The Synchronize Stacks check box enables updating of all masses simultaneously while scrolling through an image stack. Adjusting the slider (plane selector) at the bottom of any mass automatically selects the same plane for all masses.
The MIMS Data tab displays file information and meta data.
The Process tab allows the user to generate ratio images and HSI maps. Ratio and HSI images are the result of dividing one mass image by another. Masses that have similar masses will automatically show up in list form in the Process tab. Others that do not show up by default can be entered manually using the Add... button. A ratio image will appear when the user selects one (or more) in the list and clicks Display Ratio. When moving the mouse pointer over the ratio image, the status box along the lower border of the NRIMS Analysis Module window displays the raw numerator and denominator counts as well as the ratio value.
HSI images are similar to ratio images but are different in that they use a combination of the the ratio value of a pixel, the counts of one of the masses for the intensity, and a constant saturation value, to generate pixels in the RGB color space. Clicking the Display HSI will make the selected HSI image(s) appear. An example HSI image is provided in Appendix A. When displaying HSI images, the user has the option of displaying the actual ratio values (multiplied by the Ratio Scale Factor) or displaying percent turnover by selecting the Percent Turnover radio button. Percent turnover is determined by the naturally occurring ratio of that specific isotopic pair along with the maximum achievable ratio based on the experimental protocol.
The Threshold option sets the minimum number of counts in the numerator and/or denominator. Any pixels below that threshold will be ignored and appear black. The Ratio Range Min and Max values determine the range of the colormap used to display the image. The RGB Min and RGB Max values determine the intensity scale used in the image. The Transparency selects the method for computing the intensity component of the HSI image, and the Label option enables a color scale bar to be become visible. Selecting the Use Sum radio button will generate a single HSI (or ratio) image representative of the entire stack. Selecting the Use Window radio button will use the sum of both masses over a sliding window with the specified window size. This can be very useful for finding small features in large image stacks that have low counts. Also, a median filter can be applied to a ratio or HSI image by selecting the Median Filter Ratio radio button.
The Process tab is used to generate ratio and HSI images.
The Contrast tab allows the user to control the contrast settings, by adjusting the sliders, for any given mass, ratio or sum image (HSI images do not have contrast settings, instead their display parameters are controlled using the Process tab). It also displays a histogram of intensity values for any of these types of images. Clicking on an image brings it into focus and the histogram and contrast settings will update to reflect the values of the current image in focus. The histogram and contrast settings will also update by selecting the window in the combobox located at the bottom of the Contrast tab. All mass, ratio and sum images should appear in the combobox.
If changes to the default contrast settings are made, clicking the Reset button will bring those settings back to their default values. Clicking Auto will iterate through a set (usually 5 or 6) of contrast settings, eventually returning back to default values. The Set button allows the user to input values for min and max that are outside the range of those provided by the Minimum and Maximum sliders.
Mass, ratio and sum images can also be set to Auto Adjust meaning that each time a new slice is selected in the stack, new contrast settings are calculated. If Auto Adjust is not selected, the contrast settings for the first image in the stack are used throughout the stack.
The user has the additional capability of displaying the image using Lookup Tables other than the gray table used by default. There are several options for displaying the data and each image can be set independently.
The Contrast tab controls and displays contrast settings for mass, ratio and sum images.
The Stack Editing tab is reserved for functions that relate to the editing and manipulating of mass images. This includes deleting and reinserting planes, applying translation, compressing the image and generating sum images. One important thing to note is that there are two indices for an image, both of which are displayed: True index and Display index. The True index of a plane in an image never changes, but the Display index depends on what planes have been deleted. For example: entering 1-5 and clicking the Delete, then entering 6-10, and clicking Delete will -not- delete the first 10 planes of an image. Doing that is equivalent to entering 1-5, 11-15 and clicking Delete. The Reinsert button uses the indices of the original data, so entering entering 1-5, 11-15 would reinsert the previously deleted planes.
The Stack Editing tab is used for image manipulation.
A plane that is currently displayed can be translated using the Translate X or Translate Y spinners, or by entering a value in those text fields. The image can also be registered automatically by clicking on a specific mass to use and then clicking the Autotrack button. This will call the autotrack algorithm which will automatically attempt to determine the best per image translations for a best fit alignment throughout the stack. Clicking this button will open the AutoTrack Manager, which will allow the user to set a number of parameters including Autotrack all images, Autotrack subset of images, Use subregion (ROI), Normalize tracking image, Equalize tracking image, Medianize tracking image, and Show temp image. Clicking Untrack will reset all translations to zero.
The Concatenate button allows another image (or image stack) to be prepended or appended to the current image set. The Sum button will generate a sum image from whichever mass image or ratio image was most recently clicked. If the field is blank the entire image is summed, or a range of planes can be entered in the textfield (e.g. 1-20). The Compress button compresses the images into blocks of the size entered in the text field. Entering a value of 4 in the field will sum the pixel values in blocks of 4 planes, resulting in a stack of images 1/4 the original size. Any remaining planes at the end of the stack are summed together into a single block. The Uncompress button undoes the compression and restores the image to its original state - minus any translations applied and planes deleted.
Clicking the Display Action button opens a Current Action State window, displaying the changes of each plane.
Clicking on the Interleave button merges planes of data from all series of each mass into a single image stack for each mass. This is typically used with peak-switched images, in which the mass at one or more detectors is changed from one time series to the next. For masses that do not change across all series, all planes are present in the final image. E.g., if there a run with 3 time series, with 10 planes in each, the final image will contain 30 planes, arranged as:
- series 1, plane 1
- series 2, plane 1
- series 3, plane 1
- series 1, plane 2
- series 2, plane 2
- series 3, plane 2
and so on.
For images whose mass is switched from one series to the next, separate stacks are created from the different masses. Blank planes are inserted so that the number of planes in these images is the same as the number of planes in the images where peak switching does not occur. In the above example, if one of the images switches mass after the first series, 2 final 30 plane images would be created. The first image would contain stacks from the first series, alternating with blank planes:
- series 1, plane 1
- blank plane
- blank plane
- series 2, plane 2
- blank plane
- blank plane
- series 3, plane 3
and so on.
The second image would contain planes of data from the last two series, interleaved with blank planes:
- blank plane
- series 2, plane 1
- series 3, plane 1
- blank plane
- series 2, plane 2
- series 3, palne 2
- blank plane
and so on.
An example of interleaving is shown in the next two figures. In this experiment, two series of images were collected, with 10 planes per image. In the first figure, masses 25.15, 26.18, 27.22, 31.28, and 32.12 were acquired. After the first series acquisition, the second detector was tuned from mass 26.18 to mass 26.19. With interleaving, all planes for each mass are combined into a single series with 20 planes per image. Separate images are created for mass 26.18 and 26.19, and in order to have 20 images for those peak-switched masses, alternating blank planes were inserted. (second figure).
The Images from 2 series with peak switching of mass 26.18 to mass 26.19 (masses 31.28 and 32.12 not shown)
The Images after interleaving.
The Tomography tab allows the user to generate simple line plots or tables of ROI statistics through the stack. Simply select a set of ROIs from the ROI Manager, the desired statistics, and the mass, ratio and/or sum images and click Plot or Table. The Planes field allows the user to enter which planes are included in the plot (or table). If this field is left blank, all planes will be included. Checking the Append box will append the data to an existing plot or table if one exists, otherwise a new one will be created.
The Tomography tab is used to generate plots and tables of ROI statistics.
An example plot of the mean and stdev statistics for two different masses.
The table output will vary depending on the type of images for which statistics are being generating. Mass and ratio images that are more than one plane will output one row of data per plane. In the case of sum images and other images that are only one plane, one row of data will be produced per ROI. See Appendix B for a sample data output table.
The right side of the tab includes a histogram that displays the intensity values for the pixels within a given ROI. To set which ROI values are displayed by the histogram, the user only needs to scroll the mouse over the desired ROI. After a ROI has been drawn, it can be moved anywhere within the image by either dragging it with the mouse, or using the position spinners in the ROI Manager. If the Autoupdate Histogram radio button is selected, the histogram will update as the ROI is being moved by the mouse. Otherwise, it updates when the move is complete.
A line ROI represents a special case because it has no enclosed area, so its values will not be represented in the histogram. Instead, the user can select the Dynamic Profile button to generate a profile plot of the pixels which the line intersects. An example of a mass image with a line ROI and its profile plot is provided below.
A mass image with a line ROI.
A line ROI Profile Plot.
The ROI Manager (see below) gives the user functionality relating the ROIs in a given image. It is comprised of two lists, one labeled Groups and one labeled ROIs, as well as a panel on the right hand side containing buttons, spinners and checkboxes.
The ROI Manager window with several groups and ROIs, visible in the next figure.
Mass image with ROIs, 12C14N
To create a ROI, first select the type of ROI to draw by clicking one of the corresponding toolbar buttons in ImageJ’s main window (any of the five buttons on the left, i.e Rectangle, Circular, Polygon.. etc, see the top right window in the OpenMIMS desktop screenshot). Then place the mouse over the area and drag the mouse while holding the left mouse button down. When the left mouse button is released, the corresponding ROI will appear in all image windows as well as in the list of ROIs in the ROI manager. Clicking on a previously drawn ROI in ROI Manager list will automatically highlight the selected ROI in all mass, ratio, sum and HSI images.
The Groups list allows the user to organize ROIs into groups. The user can create new groups, delete existing ones, and assign ROIs into groups. An ROI can only belong to a single group. Clicking on a group will reduce the ROIs list to only those ROIs belonging to that group. Clicking on the elipses group (...) will always show all ROIs.
To create a new group, right click anywhere in the Groups list and select New group from the resulting popup menu. In the subsequent dialog (Figure 13), one can create one or more new groups, and designate the groups as segmentation training groups, if the groups will be used in segmentation.
The new ROI groups dialog.
When an ROI is created, it is placed in the currently-highlighted group. If more than one group is highlighted, the new ROI is placed in the default group.
There are 3 types of groups. Normal, Segmentation Training, and Segmentation Result. The Highlight group type combobox at the top of the ROI manager window can be used to highlight groups of a specific type. You can right-click on on selected ROIs and assign or deassign them to different groups and/or tags.
When any changes are made in the ROI Manager, such as creation of new ROIs, groups, or tags, as well as deletion of any of these items, ROI autosave is activated. After a time interval that you can change in the Preferences dialog, OpenMIMS saves the current ROIs in a hidden subdirectory (.tmp) of the directory from which the .im or .nrrd files was opened. For any given .im or .nrrd file, only the last 10 ROI files are kept. At the bottom of the ROI Manager window, the remaining time before the next autosave is displayed if any edits have been done. Right-clicking on this timer text brings up a dialog from which you can cancel the autosave countdown. When an autosave event occurs, the time at which the autosave occurred is displayed. You can also force an autosave event by right-clicking on the Last ROI autosave text, and this can be done even before the first display of the autosave time. Autosave can be effectively disabled by setting the time interval in the Preferences to a large number, such as 999,999. Changes to this time interval do not take effect until the next launch of OpenMIMS.
Delete will delete the selected ROIs in the ROI list. If none are selected then it will delete them all, after prompting the user. Rename allows the user to rename the ROI from its default name. Open will open an ROI file that had been saved from a previous session and overwrites the current list. Save will save a single ROI to a file or a group of them to a .zip file. Measure will open a statistics box that will display statistics for all ROIs for the current image. Deselect will deselect any ROIs that have been highlighted. The More>> button offers the user some more complicated features relating to the combining and splitting ROIs. ROIs can be moved on a pixel by pixel basis using the X Pos. and Y Pos. spinners. ROIs can also be moved by the user by dragging them across the image with the mouse cursor. Adjusting the Width and Height spinners will adjust those values but only for rectangular and circular ROIs.
If the user moves an ROI, it will move for all images in the stack unless the Move All checkbox is unchecked, in which it will only be moved for the current plane. The user can choose to hide all ROIs by checking the Hide All ROIs checkbox or just hide the labels by checking the Hide Labels checkbox.
Another item in the Preferences dialog controls whether or not to present the user with a dialog for controlling the loading of autosaved ROIs when the OpenMIMS application is started. The users can choose to load the last saved ROI, not load any ROI, or delete all autosaved ROIs.
The load ROIs dialog allows for loading of autosaved ROIs at application startup time.
The Segmentation tab allows the user to perform an algorithmic segmentation of a MIMS image to automatically generate ROIs for a given image plane. The algorithm used is a support vector machine (SVM) based segmentation that classifies pixels in the image using methods similar to Fuller et. al. ^[1]^. A full description of SVMs is outside the scope of this document but will be detailed in a forthcoming paper. The algorithm can use many values or features to classify a pixel: the value of that pixel in a set of mass or ratio images, the mean and standard deviation in a neighborhood around that pixel, and the gradient around that pixel. Note that the ability to use other features may be added later. To perform a segmentation the user clicks on the Config button and is presented with the SVM configuration GUI. The Export button will export the SVM and data if the SVM has been trained using the libSVM format.
*The Segmentation tab.
In the Model Configuration dialog, the user may select which mass and ratio images to use for the segmentation (segmentation can not be performed on Sum images). One can choose whether or not to use the Neighborhood parameters (mean and standard deviation) and the Gradient as features, as well as the size of the neighborhood radius. Some parameters used by the SVM library (in our case libSVM [3]) by clicking the Setup button, specifically the type of kernel to use and the level of cross validation. We recommend using the radial basis function (RBF) as the kernel. The level of cross validation may be reduced to increase speed at the possible cost of accuracy.
SVM configuration GUI.
To train the SVM the user needs to choose at least several ROIs that represent a given class of pixels. First a class is added to the Class Manager by clicking the Add button. Then the representative ROIs are drawn. Then the Sync button is clicked to sync the ROIs in the ROI Manger to the class in the Class Manager. Finally the users clicks the Ok button to complete the SVM configuration.
The SVM must be trained by clicking the Train button, located below Model description in the Segmentation tab. It should be noted that the SVM can be saved either before training (saving all parameter settings and training ROIs) or after training (also saving the trained SVM). The segmentation of the entire image can be performed by clicking Predict and the result of this prediction can be shown by clicking Display. Currently the SVM only segments 2D images. Generating ROIs from the prediction can be done by clicking ROIs which will generate a set of ROIs for each class ignoring groups of pixels of the same class that are below the value in the min area field. An example of a segmentation can be seen in Appendix C.
The Registration script allows the user to employ the ImageJ plugin TurboReg to align the open images with an image taken from an external source, for example, an electron microscope image of the same sample that was used for generating the MIMS images. Detailed information about the TurboReg plugin can be found here http://bigwww.epfl.ch/thevenaz/turboreg/
Starting the OpenMIMS Image Registration script IMPORTANT: It is strongly recommended that you compress your OpenMIMS images before use.
- Open your target image in File-¿Open Non-MIMS Image:
- Create two ROI groups in the OpenMIMS Roi Manager called ”source” and ”target”:
- Select the point ROI tool (right next to the angle tool). Use this to create 3 rois on landmarks of your choice on the non-OpenMIMS image.
- Assign these three ROI’s to the ”target” group using the ROI Manager.
- Create three ROI landmarks on your OpenMIMS images. IMPORTANT: these ROI’s must be placed in the same order and desired location as the target ROI’s i.e. the first target ROI you created will be registered to the first source ROI you create, and similarly for the second and third.
- Assign these three ROI’s to the ”source” group using the ROI Manager.
- Run this script.
- If you want to work on all the images simultaneously afterwords, use Analyze-¿Tools-¿Synchronize Windows. After registration is complete, you should see output in the Log window somewhat like that shown in Figure 17.
The MIMS Log tab keeps a record of what the user has done: e.g. deleting planes, translating planes, auto- tracking, etc. Various bits of debug data are also displayed on this tab. This information is not saved and is most likely of little use to most users.
The MIMS Log tab contains metadata and debug information.
This section provides a brief description of features provided in the menu items of the MIMS application.
Open MIMS Image : This menu item will bring up a file chooser that allows the user to select which file should be opened. The plugin can read .im files and .nrrd files ^[3]^ (It can only open the .nrrd files that were generated by the plugin). Additionally it can open any .ratio, .hsi, and .sum file that was generated by the plugin when saving from a previous session.
Open Non-MIMS Image : This menu item will bring up a file chooser that allows the user to open png, tif, and jpg images. This is useful, for example, in the registration of .nrrd and .im images with electron microscopy images.
Open Next : This menu item opens the next file within the directory of the file currently open.
Save Image : This menu item will save whatever modifications have been made to the original .im file (translations, dropped planes, etc.) into a new binary file with a .nrrd file extension. The OpenMIMS plugin is capable of reading the both .im files and the .nrrd files that it generates.
Save Session : This menu item is similar to the Save Image menu item however in this case additional files will be created for each ratio, HSI and sum image that is open at the time of saving. These files can be opened individually at a later date so long as the .nrrd file that was created with them is stored in the same directory.
Files Manager : Display the Files Manager dialog, which shows the currently-open image file, a combobox with a list of previously opened files, and radio buttons that allow the reloading of a previously opened file with its file settings, the current file settings, or the default file settings.
The Files Manager dialog.
Exit : Exits the application. ImageJ stays open as well as all opened images.
Preferences : Opens a dialog box which allows the user to set customized preferences.
Restore Mims : This menu item will reset the current image to its original state, all translations will be set back to zero and all dropped images will be reinserted. Functionally it is the same as reopening the current image.
Tile Windows : This menu item will take all of the currently open *image* windows and rearrange them in a grid on the desktop.
Close...> Close All Ratio Images: Closes all currently open ratio images. Close...> Close All HSI Images: Closes all currently open HSI images. Close...> Close All Sum Images: Closes all currently open sum images.
ROI Manager : This menu item open the ROI Manager. The ROI manager will also open whenever a ROI is drawn.
Additional menu items are located here allowing the user to view or hide individual mass images.
Generate Report : Opens a dialog box which captures the current image and has a text area that allows the users to enter notes. When the user clicks OK an .rtf formatted report is generated. Subsequent images and notes can be appended to the report. This functionality is useful for recording important information while analyzing images.
LibreOffice:
Sum All Open : Generates a sum image for all open mass and ratio images. It will not generate a sum image for open HSI images. See the Process section to see how to generate sum images for HSI images.
Import .im List : The OpenMIMS application has the ability to read a text file with a list of file names and it will open those image files, appending them to one another. *NOTE* All image files must exist in the same directory as the text file that references them. An example image list file is provided in Appendix D.
Capture Current Image : Selecting this menu item will produce a screen capture of the the last image clicked by the user (whichever image has the current focus). The new image is an RGB image of exactly what is displayed on the screen, including things like ROI outlines.
Image Notes: : Opens up a text area that allows user to enter notes regarding an image. These notes will be stored with other metadata when saving the file.
Recompute All:
Batch convert to nrrd : Allows the user to batch convert a set of .im files into .nrrd files. Can also perform tracking of the image while converting.
Find mosaic file:
Find stack file:
Center of mass autotrack:
Export-> Export...> Current Image : Export all images) as .png files.
Export-> Export All Derived (png) : Exports all derived images (ratio, HSI and sum images) as .png files.
Export...> HSI Image 3D : images) as .png files.
Generate HTML: : Closes all currently open ratio images.
Generate Table : Closes all currently open HSI images.
Generate Stack : Generates a new ratio or HSI image with independent scrollbars rather than the single plane ratio or HSI images that are generated by default.
Composite : Selecting this menu item will bring up a dialog which allows the user to quickly create a composite image with up to 4 channels. Any mass, sum, or ratio image can be used for the Red, Green, Blue, or Grey channels or a channel may be left blank. Each channel uses a simple color LUT. The min/max values for each LUT are taken from the underlying images. For example if the m26 mass image is used for the Red channel that channel will have the same min/max values. Changing the min/max values (or brightness/contrast) on the Contrast tab for the m26 image will automatically update the Red channel in the composite.
Apply dead time correction : Applies a dead time correction to the data. A 44 nanosecond correction is applied to the data.
Apply QSA correction : Applies a QSA correction to the data. Applying the QSA correction automatically applies a dead correction.
About OpenMIMS: : Applies a dead time correction to the data. A 44 nanosecond correction is applied to the data.
Documentation: : This is a link to OpenMIMS on GitHub
Sample Data : Applies a QSA correction to the data. Applying the QSA correction automatically applies a dead correction.
Appendix A
An example of an HSI image.
Appendix B
Sample output table showing mean and standard deviation for a 40 plane image file with three ROIs.
Appendix C
]
A segmented image with 9 classes and no post processing.
Appendix D
A sample image list file.
- ↑ Fuller et. al. Segmentation of Three-dimensional Retinal Image Data. IEEE Trans. Vis. Comput. Graph. 16(6):1719-1726, 2007.
- ↑ libSVM http://www.csie.ntu.edu.tw/~cjlin/libsvm
- ↑ http://teem.sourceforge.net/nrrd