# Expected Input

### Experiment Root Folder

The following is an example dataset format after formatting:

```
[2018.10.10-Hu.Tonsil.FFPE]
├[cyc001_reg001]
|   ├ 1_00001_Z001_CH1.tif
|   ├ ...
|   └ 1_00025_Z009_CH4.tif
├ ...
├[cyc008_reg001]
├ channelNames.txt
├ experiment.json
└ exposure_times.txt

[] = Folders
```

![Hu Tonsil FFPE / cyc005\_reg001 / 1\_00001\_Z005\_CH1.tif (DAPI)](https://1268872846-files.gitbook.io/~/files/v0/b/gitbook-legacy-files/o/assets%2F-LWT7bo4Xbl2jOnmH1yB%2F-L_9hujx5DhG5_74cqK8%2F-L_A3xVTBhqJUp9WNoLS%2FHu_Tonsil_FFPE_1_00001_Z005_CH1.png?alt=media\&token=9724e13b-e305-4fc8-bb59-70956cf51604)

### Cycle-Region Folders

The top folder is the base experiment folder, encapsulating all data for a single experiment. The number of cycle-region folders should match the expected number of cycles and regions imaged. The name of the cycle-region folders may vary (e.g. 'Cyc1\_reg1' or 'cyc001\_reg001' or 'cyc001\_reg001\_\<timestamp>').

Each cycle folder should contain images of equal size. Keyence images are 1920x1440, while Leica and Zeiss are 2048x2048. The number of images must match the expected region size (e.g. 900 images = 5 region-width x 5 region-height x 9 z-slices x 4 channels). For Keyence datasets, there will be a .bcf file for each cycle folder (i.e. 901 total files).

Naming scheme is:

```
1_00001_Z001_CH1.tif
|   |     |   |
|   |     |   └ Channel number with 'CH' prefix
|   |     └ Z-slice number, 3-digit padding with 'Z' prefix
|   └ Tile number, 5-digit padding
└ Region number, no padding

All numbers are 1-index.
```

### Experiment JSON File

Experiment parameters file (experiment.json) should be generated by CIM. If unavailable, the file will be automatically generated by the processor using Keyence default values. Before starting a processing run, please check the experiment and processing parameters, especially region size and tile overlap. For Leica and Zeiss users, please double-check the image parameters, including numerical aperture and pixel resolution.

### Channel Names Text File

Channel names text file (channelNames.txt) should contain a biomarker name for every channel and cycle. Blank cycles to be used by background subtraction should be named 'Blank'. Empty cycles to be ignored by background subtraction should be named 'Empty'.

> DAPI-1\
> Blank\
> Blank\
> Blank\
> DAPI-2\
> CD15\
> Ki67\
> CD45\
> ...\
> DAPI-7\
> CD8\
> Empty\
> CD11c\
> DAPI-8\
> Blank\
> Blank\
> Blank

### Exposure Times Text File

At least one minimum exposure blank cycle is required for background subtraction. A corresponding exposure times file (exposure\_times.txt) is also required. The exposure times file should contain a header followed by
&#x20;exposure times for each cycle (e.g. '10' or '1/100s') deliminated by tabs or commas. For additional information on background subtraction modes, please see [here](background-subtraction).

| CYC | CH1 | CH2 | CH3 | CH4 |
| --- | --- | --- | --- | --- |
| 1   | 10  | 500 | 350 | 500 |
| 2   | 10  | 500 | 350 | 500 |
| ... | ... | ... | ... | ... |
| 8   | 10  | 500 | 350 | 500 |





