How do I handle multi-channel images?

How do I handle multi-channel images?#

What will I learn?

  • How to re-use the same display settings across multiple images

  • How to detect nuclei in fluorescence images

  • How to use the channel viewer to make sense of images with many channels

QuPath can handle multiplex images with quite a lot of channels (we’ve tried up to 70).

This page introduces some useful tools for working with multi-channel images.

Display settings & multiplexed images#

What to do

  1. Open LuCa-7color_[13860,52919]_1x1.tif.

    • Choose Fluorescence as the image type

  2. Open the brightness and contrast dialog ../../_images/CONTRAST.png.

  3. Adjust the maximum value for the DAPI channel so that nuclear staining is more obvious.

  4. Enter DAPI into the Settings dropdown box

  5. Click Save.

In “How do I change colours?” we explored image visualization settings in the brightness and contrast dialog.

QuPath makes a “best guess” at what will produce a useful visual representation of each image but this isn’t always optimal. It can also result in variability in how images appear across images in a project.

To ensure similar images use the same display settings, you can select the Apply to similar images checkbox when opening similar images within the same project. Alternatively, you can save your display settings as a viewing profile.

This is useful if you have a collection of images that you want to view with the same brightness and contrast settings, particularly if there are many channels to adjust.

Note

Viewing profiles can only be applied to images with the same number of channels, and only if the channels have the same names.

Detecting cells#

What to do

  1. Create an annotation using your favourite annotation tool.

  2. Run Analyze ‣ Cell detection ‣ Cell detection…

  3. Without changing any parameters, click Run.

  4. Were any cells detected…?

QuPath aims to make cell detection straightforward, with sensible default parameters… although that doesn’t always work.

In this case, the threshold that QuPath used is very far off – and so no cells were found.

You can figure out a better value by moving the cursor over nuclei in the viewer and looking at the pixel values displayed in the bottom right of the viewer. Let’s try again with better settings.

What to do

  1. Enter 1 in the Threshold field.

  2. Click Run.

  3. Inspect the results.

    • Load the DAPI display settings you saved earlier, and see how well the nuclear segmentation matches what you can see in the DAPI channel.

Channel viewer#

What to do

  1. Select from the menu View ‣ Show channel viewer.

  2. Move the cursor over the image to view the channels in the image.

  3. Toggle the visibility of one or more channels.

    • You can use the brightness & contrast window, or type a channel number

  4. Right-click on the channel viewer and choose Zoom… ‣ 400% to see more detail

QuPath includes a useful channel viewer for visualizing different channels of an image simultaneously – without needing to toggle each channel one-by-one.

The channel viewer can also adapt whenever channels are toggled, or brightness & contrast settings adjusted.

../../_images/channel_viewer.png

Fig. 48 Multiplex image with the channel viewer open#

Detection channels#

QuPath will try to guess which channel should be used to detect nuclei based on channel names and commonly-used nuclear stains (e.g., DAPI, Hoechst). However, sometimes you may need to choose the detection channel yourself.

What to do

  1. Select another channel under Detection channel (for example, FoxP3).

  2. Click Run.

  3. Inspect the results. How many cells are detected using this channel?

Contents