geomx dsp imagej guidelines -...

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.

GeoMx DSP ImageJ Guidelines PMR-10198

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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.

PMR-10198 GeoMx DSP ImageJ Guidelines

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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.

PMR-10198 GeoMx DSP ImageJ Guidelines

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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.

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