geomx dsp imagej guidelines -...
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PMR-10198
GeoMx DSP ImageJ Guidelines
FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.
© 2018 NanoString Technologies, Inc. All rights reserved.
NanoString, NanoString Technologies, the NanoString logo, nCounter, and GeoMx are trademarks or registered
trademarks of NanoString Technologies, Inc., in the United States and/or other countries.
GeoMx DSP ImageJ Guidelines
The GeoMx DSP Scan Workspace allows users to
export regions of interest (ROIs) to create
custom masks in the external program of their
choice. Users may then import the segmented
images back to the DSP system and continue the
workflow. This is an optional process, available
to users who require customization beyond that
offered in the GeoMx DSP Scan Workspace.
ImageJ is one commonly-used open-source
image processing application and Fiji (Fiji Is Just
ImageJ) is another version of this program.
These guidelines describe a model process for
creating custom masks in ImageJ (or Fiji).
For instructions on exporting individual ROIs, see the ROI Selection & Collection topic in the GeoMx DSP
Instrument (Data Collection) User Manual or the GeoMx DSP Online User Manual; the latter is accessible
from the help icon in the GeoMx DSP Control Center. Note: You may alternatively segment ROIs within the
GeoMx DSP Scan Workspace; for more information on this process, see the GeoMx DSP Auto-
Segmentation Guidelines, accessible from the GeoMx DSP Online User Manual.
1. Open ImageJ.
2. Transfer your images. In a separate window,
navigate to your images exported from DSP. Select
one or more ROI to open. Drag your selection and
drop it on the ImageJ header. This will open one
image for each ROI; each image will be a stack of
single-channel black- and-white images, with each
channel map- ping to a single channel in the
florescence image. Use the scroll bar at the
bottom of each window to scroll through each
image in the stack.
Figure 1: Fiji user interface.
Figure 2: Transfer images to Fiji.
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3. Separate the stacked Tiff into single channels. From
the Image Menu, highlight Stacks, then select Stack
to Images. This will separate the stacks, so you have
an individual window for each single-channel black-
and-white image. Each window is numbered 1-4, to
help you differentiate.
4. Adjust the threshold of each single-channel image.
Select the image of interest. From the Image
Menu, highlight Adjust, then select Threshold.
• Use the sliders to isolate areas of surface
marker intensity. Consider the following
settings:
o Keep the lower bound slider at zero (left).
o Slide the upper bound slider to the right
until you have a sufficiently clean
population.
o The area represented between these two
selections will be black; moving the upper
bound to the right results in a more
conservative selection.
Figure 3: Separate stacked Tiff into single channels.
Figure 4: Adjust the threshold.
• Auto-thresholding algorithms are available in the dropdown. These can provide a starting
point but may not be appropriate or helpful, depending on the tissue and chemistry. Select
the Auto button to use the selected algorithm.
• Colors are available in the dropdown.
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• Leave the Dark background and Stack histogram boxes unchecked.
• Select the Reset button to revert to the starting image.
Select Apply. Close the Threshold window.
5. Invert to create a mask. From the Edit menu,
select Invert.
This step is important to convert the image into
the format which the GeoMx DSP System will
recognize.
• Set non-zero to collected.
• Set zero to not collected.
Figure 5: Invert to create mask.
6. Filter out noise. From the Analyze menu, select Analyze Particles.
• To show a mask of particles
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• A new window will open, labeled Mask of…
followed by your source image name and the
channel number (0001, 0002, etc.). Select the
mask image. From the Edit menu, highlight
Selection, then select Create Selection.
• Select the original thresholded binary image.
From the Edit menu, highlight Selection, then
select Restore Selection.
• From the Edit menu, select Invert. Click
somewhere on the image to deselect the small
particles.
Figure 7: Create selection.
Figure 8: Restore selection and invert.
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7. Remove nuclei holes:
• From the Edit menu, select Invert.
• From the Analyze menu, select Analyze
Particles. To show a mask of particles
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9. (Optional Step) Smooth erode the dark. From the Process menu, highlight Binary and select Erode.
10. Save. From the File menu, select Save As, and select .png.
For instructions on importing individual ROIs, see the ROI Selection & Collection topic in the GeoMx DSP
Instrument (Data Collection) User Manual or the GeoMx DSP Online User Manual; the latter is accessible
from the help icon in the GeoMx DSP Control Center.
For additional ImageJ resources, visit:
• Imagej.nih.gov/ij/docs/index.html
• Imagej.nih.gov/ij/developer/macro/functions.html
IMPORTANT: Use caution with erode-dilate functions, as one does not act as
an undo function for the other.