Imaging Acquisition Guidelines

 In Situ Sequencing Kit

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Microscope set-up

For visualisation of CARTANA in situ sequencing data, we recommend the following microscope and computer requirements:

  • Epifluorescence microscope with autofocus function
  • Automated (x, y, z) stage with the stage controller
  • Light source and filter cubes for optimal imaging of DAPI (358/461), Alexa Fluor® 488 (495/519), Cy3 (550/570), Cy5 (650/670) and Alexa Fluor® 750 (749/775)
  • Objectives 4x, 20x and 40x
  • Digital camera (pixel size 5-7 μm)
  • PC and data storage with specifications suitable for imaging and processing data ranging from 4TB to 60TB

General acquisition settings

Due to the typical size of ISS spots (< 1 µm), all imaging must be performed with a 20x N.A.0.75 objective or greater. This is necessary as the RCPs will not be properly resolved otherwise.

  • 5 channels need to be imaged:
    • DAPI (or nuclear stain channel) – this is to assess the density of cells within the imaged area.
    •  Alexa Fluor® 488, Cy3, Cy5 and Alexa Fluor® 750 – these are the channels in which ISS spots can be visualised.
      • Many tissues exhibit background fluorescence in multiple channels that can be mistaken for ISS spots. The actual ISS spots are always visible in only 1 channel.
      • In any channel, saturation of the fluorescent signal should be avoided.
  •  Where possible, z-stacks should be used to cover ISS spots in three dimensions:
      • Use z-stacks to cover the whole thickness of each section. For example, a 10 μm section could have 5 steps in each direction from center. The size of the z-stack steps should not exceed 0.9 μm.


CARTANA 20x images using DAPI and Cy5 channels

Figure 1: 20x images using DAPI and Cy5 channels.

Figure 1 shows 20x images of a tissue section. The nuclei are visualised in the DAPI channel and the ISS spots are visualised in the Cy5 channel. The white square in each image is the area shown in the close-ups.

Data Processing

The raw microscope images need to be pre-processed before image analysis.

  • If z-stacks were taken (highly recommended), perform a maximum intensity projection.
  • If several multipoints are imaged to cover the same section, perform stitching to combine them.
  • Export images in a TIFF file format
    • Each channel should be a separate TIFF file (i.e., DAPI, FITC, Cy3, etc.)
    • The TIFF files should be 8 or 16-bit grayscale images