Input Files

HCR Files

Create an experiment directory in the following format:

YYYY-MM-DD_EXPERIMENT

Examples:

2024-12-20_FN-SNR-8
2025-01-06_XII-2
2023-06-08_MC-SC-5

Create a subdirectory within this folder for each round (pre-imaging round number is 0) in the following format:

YYYY-MM-DD_r#_HCR

Examples:

2024-12-23_r1_HCR
2025-01-06_r0_HCR
2023-06-14_r2_HCR

The HCR tag is required for HCRprocess to recognize the folder as a round folder. Within each round folder, place your nd2 files in the following format:

UNIQUE-ID__gene1chan#_gene2chan#_gene3chan#_gene4chan#__HCR-EXTRA-INFO

Examples:

s04__eGFP488_dTom546_NT647__HCR-40x.nd2
s03-left__Phox2b594_Snap25488_Calb1546_Slc32a1647__HCR-40x-zstack.nd2
s01-ant__Phox2b594_Snap25488_Calb1546_Slc32a1647__HCR.nd2

Note the double underscores separating both the unique identifier from channels and channels from extra information. Make sure a single underscore separates each channel, and that channels are listed in the order they appear in the file (the order in which they are acquired in NIS-Elements). The unique identifier and extra information can be whatever you want, but don’t include underscores in them. The HCR tag is required for HCRprocess to recognize a file as an HCR image file that requires processing. Here’s an example of what a full experiment directory might look like, before any processing in HCRprocess:

2024-11-10_MC-SC-4
|  2024-11-10_r0_HCR
|  |  s01-20x-overview.nd2*
|  |  s02-20x-overview.nd2*
|  |  s01-ROI.png*
|  |  s02-ROI.png*
|  |  s01__eGFP488_dTom546_NT647__HCR.nd2
|  |  s02__eGFP488_dTom546_NT647__HCR.nd2
|  2024-11-13_r1_HCR
|  |  s01-20x-overview.nd2*
|  |  s02-20x-overview.nd2*
|  |  s01__Phox2b594_Snap25488_Calb1546_Slc32a1647__HCR-zstack.nd2
|  |  s02__Phox2b594_Snap25488_Calb1546_Slc32a1647__HCR-zstack.nd2
|  2024-11-16_r2_HCR
|  |  s01-20x-overview.nd2*
|  |  s02-20x-overview.nd2*
|  |  s01__Nr4a2594_Snap25488_Zfhx4546_Ebf3647__HCR-40x.nd2
|  |  s02__Nr4a2594_Snap25488_Zfhx4546_Ebf3647__HCR-40x.nd2

= ignored by automatic file detection

Histology Files

HCRprocess can also autodetect and process histology image files. First add a histology subdirectory in the experiment folder using the following formatting:

YYYY-MM-DD_histo

The histo tag is required for HCRprocess to recognize the folder as a histology folder. Within the histology folder, place your image files in the following format:

REGION-#__fluorophore1chan#_fluorophore2chan#_fluorophore3chan#__histo-extra-info

Examples:

MC-2__dTomTRITC_NTCy5__histo.vsi
SC-5__eGFPFITC_dTomTRITC_NTCy5__histo-10x.vsi
FN-1__eGFPFITC_NTCy5__histo.nd2

Make sure a single underscore separates each channel, and that channels are listed in the order they appear in the file (the order in which they are acquired). The histo tag is required for HCRprocess to recognize a file as a histology image file that requires processing. Currently HCRprocess can automatically recognize and process histology image files of the following formats: vsi, nd2.