Full Experiment Analysis Workflow

Initialize datastore

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flowchart TD
 subgraph s1["Define datastore"]
        n1["Number of tiles"]
        n2["Microscope metadata"]
        n3["Experiment metadata"]
        n4["qi2labdatastore"]
  end
    n1 --> n4
    n2 --> n4
    n3 --> n4

    n1@{ shape: notch-rect}
    n2@{ shape: notch-rect}
    n3@{ shape: notch-rect}
    n4@{ shape: lin-cyl}

Populate datastore

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flowchart TD
 subgraph s1["Place raw data into datastore"]
        n5["Raw data"]
        n12["Camera correction"]
        n6["Geometric transformation
        (local to global)"]
        n7["Experiment order"]
        n8["Fidicual data"]
        n9["MERFISH data"]
        n10["Global tile positions"]
        n11["qi2labdatastore"]
 end
    n5 --> n12
    n12 --> n6
    n6 --> n7
    n7 --> n8
    n7 --> n9
    n8 --> n11
    n9 --> n11
    n10 --> n11

    n5@{ shape: procs}
    n6@{ shape: notch-rect}
    n7@{ shape: notch-rect}
    n8@{ shape: procs}
    n9@{ shape: procs}
    n10@{ shape: procs}
    n11@{ shape: lin-cyl}
    n12@{ shape: notch-rect}

Deconvolve, locally register, and spot predict all tiles

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flowchart TD
 subgraph s2["MERFISH preprocessing"]
        n17["Deconvolution"]
        n18["U-FISH prediction"]
        n19["Tile warping"]
  end
 subgraph s3["Fiducial preprocessing"]
        n14["Deconvolution"]
        n15["Rigid registration"]
        n16["Deformable registration"]
  end
 subgraph s1["Local preprocessing"]
        s2
        s3
        n1["Local tile registrations back to round 1"]
  end
  n13["qi2labdatastore"]

    s3 --> n1
    n1 --> s2
    s1 <--> n13
    n14 --> n15
    n15 --> n16
    n17 --> n18
    n18 --> n19
    n1@{ shape: notch-rect}
    n13@{ shape: lin-cyl}
    n14@{ shape: procs}
    n15@{ shape: procs}
    n16@{ shape: procs}
    n17@{ shape: procs}
    n18@{ shape: procs}
    n19@{ shape: procs}

Global registration and fusion of first fiducial round

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flowchart TD
 subgraph s1["Global registration"]
    n3["Fiducial data"]
    n4["Multiview-Stitcher"]
 end
 n1["qi2labdatastore"]
 n2["XY downsampled, Z max projected, and fused global fiducial image"]
 n5["Cellpose"]
 n6["2D cell segmentations"]
 n7["Optimized global tile positions"]
 n8["User optimized parameters"]

    s1 <--> n7
    n7 <--> n1
    s1 --> n2
    n3 <--> n4
    n2 --> n5
    n5 --> n6
    n6 --> n1
    n8 --> n5
    n1@{ shape: lin-cyl}
    n2@{ shape: procs}
    n3@{ shape: procs}
    n6@{ shape: procs}
    n7@{ shape: notch-rect}
    n8@{ shape: notch-rect}

Pixel decoding

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flowchart TD
 subgraph s1["Pixel decoding"]
    n2["MERFISH data"]
    n3["Global normalization estimate"]
    n4["Iterative normalization estimate"]
    n5["Pixel decoding"]
    n6["False-positive filtering"]
    n7["Overlap cleanup"]
    n8["Cell assignment"]
    n9["Data prep for resegmentation"]
 end
 n1["qi2labdatastore"]

    s1 <--> n1
    n2 --> n3
    n3 --> n4
    n4 --> n5
    n5 --> n6
    n6 --> n7
    n7 --> n8
    n8 --> n9

    n1@{ shape: lin-cyl}
    n2@{ shape: procs}
    n3@{ shape: procs}
    n4@{ shape: procs}
    n5@{ shape: procs}
    n6@{ shape: procs}
    n7@{ shape: procs}
    n8@{ shape: procs}
    n9@{ shape: procs}

3D segmentation based on decoded RNA

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flowchart TD
 subgraph s1["3D segmentation"]
    n3["Decoded, cell-assigned RNA"]
    n4["Baysor"]
    n6["User optimized parameters"]
 end
 n1["qi2labdatastore"]
 n2["3D segmentations"]
 n5["Updated RNA assignments"]

    s1 <--> n1
    n3 --> n4
    n6 --> n4
    s1 --> n2
    n2 --> n5
    n5 --> n1
    n1@{ shape: lin-cyl}
    n2@{ shape: procs}
    n3@{ shape: procs}
    n5@{ shape: procs}
    n6@{ shape: notch-rect}