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Microscope Assessment

Before processing images for the Illumina Spatial Transcriptome Assay, the Optical Target Test should be performed using the Illumina Bright Field (BF) Optical Target.

The Optical Target Test provides the following key metrics:

  • Estimated pixel size (microns) of the camera

  • Full Width Half Maximum (FWHM) for assessing focus quality

  • Shading uniformity observed in the FOV

  • An optical distortion heatmap

The Illumina BF Optical Target is a 75 mm x 25 mm x 1 mm slide featuring patterned elements in the center. This test must be conducted using the 20X objective lens and the specific camera intended to be used with the Illumina Spatial Transcriptome Assay. The microscope assessment includes evaluation of focus quality, detection of stage tilt, analysis of optical distortion, and assessment of shading uniformity. These metrics are valuable for diagnosing and troubleshooting image quality issues.

To begin the Optical Target Test assessment workflow, open the Illumina Spatial Image Tool (ISIT) and select Assess microscope.

Inputs

To configure the Optical Target Test in the Illumina Spatial Image Tool, provide the following information:

  • Microscope Name

    • Enter the name or identifier to be used when saving your microscope profile.

  • Z-Stack Step Size

    • The step size value used during imaging (e.g., 0.2 μm, 0.3 μm, or 0.4 μm).

  • Fiducial Images Folder

    • This folder contains the focused field-of-view (FOV) Z-stack images of the concentric circles on the Illumina BF Optical Target taken during the imaging portion of the Optical Target Test.

  • Pinhole Images Folder

    • This folder contains the FOV Z-stack images of the white-dot-in-black-background pattern region of the Illumina BF Optical Target taken during the imaging portion of the Optical Target Test.

  • Empty Tile Image

    • This image is of the blank region of the Illumina BF Optical Target taken during the imaging portion of the Optical Target Test.

Screenshot of the initial page of the Microscope Assessment workflow

Outputs

The Optical Target Test provides the following key metrics:

  • Estimated pixel size (microns) of the camera

  • Full Width Half Maximum (FWHM) for assessing focus quality

  • Shading uniformity observed in the FOV

  • An optical distortion heatmap

Save the estimated pixel size with the save button for use in the image processing workflow.

Take a screenshot of the output screen, share it with your Illumina representative, and save a copy for future troubleshooting if needed.

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