CycloBranch
CrossVis

CrossVis

The tool is opened using the command "Tools -> CrossVis" from the Main Window. The function is available if the imaging mass spectra are processed (*.imzML file). Use F1 to open this help.

crossvis.png
CrossVis

Toolbar

Open Image (CTRL + O)

Open an optical/histology image.

Save Image (CTRL + S)

Save the modified image.

Close (Esc)

Close window.

Show Selection (Ctrl + E)

Show current selection rectangle.

Color Scale and Ruler (CTRL + C)

Show/Hide color scale and ruler.

Absolute Intensity (CTRL + I)

View absolute/relative intensities of peaks.

Zoom In (CTRL +; mouse wheel up)

Zoom in.

Zoom Out (CTRL -; mouse wheel down)

Zoom out.

Zoom Reset (CTRL + R; mouse wheel pressed)

Reset zoom.

Ruler Size

The current size of ruler [micrometers].

HTML Documentation (F1)

Show HTML documentation.

Layers

The list of layers is on the right side of the CrossVis tool. The radio button is used to select an active layer. The checkbox is used to show/hide a layer. The slider and the number in the first spin box is the opacity of a layer (0 = layer is not visible; 100 = layer is not transparent). The second spin box is the order of a layer (z-value).

Each layer has an associated toolbar. The toolbar associated with a layer is green if the corresponding layer is selected (i.e., the corresponding radiobutton is checked). The green values can be also modified by mouse selection.

Compounds

The range of X and Y coordinates to be selected. The button 'Select Region' applies the changes (Enter key can also be used if a corresponding text field is focused). The 'Reset Region' button clears the selection. The selection can also be performed using mouse. To draw a rectangle, press and hold the left mouse button. Draw a rectangle as shown in the following figure and then release the left mouse button. You can click on the right mouse button during the rectangle drawing to cancel the selection. The size of the region can be adjusted and the region can be moved by mouse.

The number of selected points and the minimum bounding region of the selection is reported under the image. The selection of X and Y intervals automatically filters rows in the Summary Table of Matched Peaks. If a compound was filtered by its name or m/z value in Summary Table of Matched Peaks, the filtered text is shown. Both selection methods can be used together.

The standard HSL (hue, saturation, lightness) color model is used to visualize the points. The most intense point in a selected region is red and minimum intensity points are violet. A color represents a sum of relative/absolute intensities of matched peaks in a single spectrum; or the intensity of a specific compound if it was filtered by its name or m/z value in the Summary Table of Matched Peaks.

crossvis-compounds.png
CrossVis - selection of compounds

Optical Image

The toolbar is used to correlate the measured region of compounds with optical image (see below for vendor specific details). The region can also be adjusted or moved by mouse as stated above.

  • 'Horizontal Shift' - horizontal shift of points between measured area and the optical image [number of points]
  • 'Vertical Shift' - vertical shift of points between measured area and the optical image [number of points]
  • 'Max X' and 'Max Y' - the [X,Y] coordinates of the bottom-right corner of an optical image [number of points] (see below for vendor specific details)
  • 'Correlate' button (or Enter key if 'Horizontal Shift', 'Vertical Shift', 'Max X' or 'Max Y' are focused) - correlate the measured region of compounds with optical image
  • 'Default' - use the default values from input imzML file (see below for vendor specific details)
crossvis-correlation.png
CrossVis - correlation of imzML exported by FlexImaging 4.1 with optical image

Histology Image

The toolbar is used to correlate the histology image with an optical image. Then the identified compounds are visualized in histology image. The size and position of histology image can also be adjusted by mouse.

Flip Histology Horizontally (Ctrl + H)

Flip histology image horizontally.

Flip Histology Vertically (Ctrl + V)

Flip histology image vertically.

  • 'X' - horizontal position of histology image [pixels]
  • 'Y' - vertical position of histology image [pixels]
  • 'Width' - width of histology image [pixels]
  • 'Height' - height of histology image [pixels]
  • 'Angle' - rotation angle of histology image [degrees]
crossvis-histology.png
CrossVis - positioning of histology image with an optical image

Microscopy Image

The toolbar is used to correlate a microscopy image with an optical image. Then the identified compounds are visualized in microscopy image. The toolbar is associated with one navigation microscopy image "Microscopy image (nav.)" and up to ten additional non-navigation microscopy images. The values of X, Y, Width, Height, and Angle correspond to an active microscopy image which is currently selected in the list of layers on the right side.

  • 'X' - horizontal position of microscopy image [micrometers]
  • 'Y' - vertical position of microscopy image [micrometers]
  • 'Width' - width of microscopy image [micrometers]
  • 'Height' - height of microscopy image [micrometers]
  • 'Angle' - rotation angle of microscopy image [degrees]
  • 'Default' - use the default values from the TIFF file
  • 'Go' - go to the coordinates of microscopy image

It is expected that an input image was generated in TIFF file format by FEI Nova NanoSEM 450. Then the size of image in micrometers is calculated automatically from metadata stored in the input image file. If a different image was used the size of the image must be adjusted manually. The size and position of microscopy image can also be adjusted by mouse.

It is expected that the microscopy navigation image has a small maginification (e.g., 50x) and that non-navigation images have bigger magnification. The size of the image in micrometers is adjusted automatically if the input TIFF file contains corresponding metadata. In case of a microscopy navigation image, the size is adjusted automatically but the position in the optical image must be adjusted manually. In case of a microscopy non-navigation image, both - the size and the position are adjusted automatically.

Vendor-specific Details

imzML exported from FlexImaging 4.1 (Bruker)

The correct values of 'Max X' and 'Max Y' must be determined by hand as they are not included in the imzML file. Open the dataset in FlexImaging 4.1 and determine the coordinates of the bottom-right corner of an optical image as shown in the following image. R00X451Y321 means that Max X = 451 and Max Y = 321.

regions.png
Coordinates of the bottom-right image corner.

For the above example, the correct settings are

  • Horizontal Shift = 0
  • Vertical Shift = 0
  • Max X = 451
  • Max Y = 321

imzML exported from HDImaging v1.4 (Waters)

In HDImaging v1.4 click on the 'Acquire' tab, then open a pattern definition metafile (*.pdm). Click on the measured region and then click on the 'Export' icon. In the image export dialog, check the option 'Crop to selection' and uncheck the option 'Overlay regions'. Then click 'Export' and save the file. Hint: Enlarge the main window of HDImaging as much as possible to make the exported image bigger.

The correct values of 'Max X' and 'Max Y' are determined automatically from the imzML file. It may be necessary to modifify the values of 'Horizontal Shift' and 'Vertical Shift' manually to correlate the points with the optical image correctly.