finest 0.1.3

FineST: Fine-grained Spatial Transcriptomic

This software package implements FineST (Fine-grained Spatial Transcriptomics), which identifies super-resolved ligand-receptor interactions with spatial co-expression refining spot to sub-spot or single-cell resolution.

FineST comprises three components (Training-Imputation-Discovery) after HE image feature extraction:

• Step0: HE image feature extraction

• Step1: Training FineST on the within spots

• Step2: Super-resolution spatial RNA-seq imputation at sub-spot or single-cell level

• Step3: Fast fine-grained ligand-receptor pair and cell-cell communication pattern discovery

Installation using Conda

git clone https://github.com/StatBiomed/FineST.git
conda create --name FineST python=3.8
conda activate FineST
cd FineST
pip install -r requirements.txt

Verify the installation using the following command:

python
>>> import torch
>>> print(torch.__version__)
2.1.2+cu121 (or your installed version)
>>> print(torch.cuda.is_available())
True

FineST package is available through PyPI.

pip install -U FineST

## Alternatively, install from GitHub for latest version:
pip install -U git+https://github.com/StatBiomed/FineST

The FineST conda environment can be used for the following Tutorial by:

python -m pip install ipykernel
python -m ipykernel install --user --name=FineST

Tutorial notebooks:

• NPC_Train_Impute_demo.ipynb (using Virchow2; requires Hugging Face token, approval may take days)

• NPC_Train_Impute_demo_HIPT.ipynb (using HIPT; recommended for quick start)

ROI selection via Napair

To analyze a specific region of interest (ROI), use napari to select the region:

from PIL import Image
Image.MAX_IMAGE_PIXELS = None
import matplotlib.pyplot as plt
import napari

image = plt.imread("FineST_tutorial_data/20210809-C-AH4199551.tif")
viewer = napari.view_image(image, channel_axis=2, ndisplay=2)
napari.run()

Quick guide:

• A shapes layer is automatically added when opening napari

• Use the Add Polygons tool to draw ROI(s) on the HE image

• Optionally rename the ROI layer for clarity

For detailed instructions and ROI extraction using fst.crop_img_adata(), see the tutorial or video guide.

Get Started for Visium or Visium HD data

The tutorial includes:

• Visium: 10x Visium human nasopharyngeal carcinoma (NPC) data

• Visium HD: 10x Visium HD human colorectal cancer (CRC) data (16-um bin) [Sample P2 CRC]

Data Download

Download Visum FineST_tutorial_data from Google Drive or via command line:

python -m pip install gdown
gdown --folder https://drive.google.com/drive/folders/1rZ235pexAMVvRzbVZt1ONOu7Dcuqz5BD?usp=drive_link

Fast Run for Demo

bash test_demo.sh

• Note: The demo uses HIPT for image features, which is faster and doesn’t need Hugging Face token.

• For using Virchow2 (may require a token and take longer; in paper), see the detailed manual below.

• The demo uses the Visium NPC dataset; for Visium HD CRC data, follow the manual for Step 0-1-2.

• The demo runs Step 0-1; for Step 2, plese replace the trained weight_save_path with your own.

Step0: HE image feature extraction

• For Visium data, extract image features for both within-spots and between-spots.

• For Visium HD data, extract features directly from continuous squares.

Option A: Extract image features for within-spots (Visium)

For Visium (55um spot diameter, 100um center-to-center distance), extract image features of original (within) spots:

## Option A: Using HIPT (recommended for quick start, no token required)
python ./demo/Image_feature_extraction.py \
   --dataset NPC \
   --position_path FineST_tutorial_data/spatial/tissue_positions_list.csv \
   --rawimage_path FineST_tutorial_data/20210809-C-AH4199551.tif \
   --scale_image False \
   --method HIPT \
   --patch_size 64 \
   --output_img FineST_tutorial_data/ImgEmbeddings/pth_64_16_image \
   --output_pth FineST_tutorial_data/ImgEmbeddings/pth_64_16 \
   --logging FineST_tutorial_data/ImgEmbeddings/Logging/ \
   --scale 0.5  # default is 0.5

## Option B: Using Virchow2 (requires Hugging Face token)
python ./demo/Image_feature_extraction.py \
   --dataset NPC \
   --position_path FineST_tutorial_data/spatial/tissue_positions_list.csv \
   --rawimage_path FineST_tutorial_data/20210809-C-AH4199551.tif \
   --scale_image False \
   --method Virchow2 \
   --patch_size 112 \
   --output_img FineST_tutorial_data/ImgEmbeddings/pth_112_14_image \
   --output_pth FineST_tutorial_data/ImgEmbeddings/pth_112_14 \
   --logging FineST_tutorial_data/ImgEmbeddings/Logging/ \
   --scale 0.5  # default is 0.5

Option B: Extract image features for bin-squares (Visium HD)

For Visium HD (continuous squares without gaps), extract image features directly:

python ./demo/Image_feature_extraction.py \
   --dataset HD_CRC_16um \
   --position_path ./Dataset/CRC/square_016um/tissue_positions.parquet \
   --rawimage_path ./Dataset/CRC/square_016um/Visium_HD_Human_Colon_Cancer_tissue_image.btf \
   --scale_image True \
   --method Virchow2 \
   --output_img ./Dataset/CRC/HIPT/HD_CRC_16um_pth_28_14_image \
   --output_pth ./Dataset/CRC/HIPT/HD_CRC_16um_pth_28_14 \
   --patch_size 28 \
   --logging ./Logging/HIPT_HD_CRC_16um/ \
   --scale 0.5  # default is 0.5

Note: Visium HD uses .parquet for positions and .btf for images, while Visium uses .csv and .tif.

Step1: Training FineST on the within spots

Option A: Visium

Train FineST model on within-spots to learn the mapping from image features to gene expression.

## HIPT with Visium16 (patch_size=64)
python ./demo/Step1_FineST_train_infer.py \
   --system_path '/home/lingyu/ssd/Python/FineST/FineST/' \
   --parame_path 'parameter/parameters_NPC_HIPT.json' \
   --dataset_class 'Visium16' \
   --image_class 'HIPT' \
   --gene_selected 'CD70' \
   --LRgene_path 'FineST/datasets/LR_gene/LRgene_CellChatDB_baseline_human.csv' \
   --visium_path 'FineST_tutorial_data/spatial/tissue_positions_list.csv' \
   --image_embed_path 'FineST_tutorial_data/ImgEmbeddings/pth_64_16' \
   --spatial_pos_path 'FineST_tutorial_data/OrderData/position_order.csv' \
   --reduced_mtx_path 'FineST_tutorial_data/OrderData/matrix_order.npy' \
   --figure_save_path 'FineST_tutorial_data/Figures/' \
   --save_data_path 'FineST_tutorial_data/SaveData/' \
   --patch_size 64 \
   --weight_w 0.5

## Virchow2 with Visium64 (patch_size=112)
python ./demo/Step1_FineST_train_infer.py \
   --system_path '/home/lingyu/ssd/Python/FineST_submit/FineST/' \
   --parame_path 'FineST_tutorial_data/parameter/parameters_NPC_virchow2.json' \
   --dataset_class 'Visium64' \
   --image_class 'Virchow2' \
   --gene_selected 'CD70' \
   --LRgene_path 'FineST_tutorial_data/LRgene/LRgene_CellChatDB_baseline.csv' \
   --visium_path 'FineST_tutorial_data/spatial/tissue_positions_list.csv' \
   --image_embed_path 'FineST_tutorial_data/ImgEmbeddings/pth_112_14' \
   --spatial_pos_path 'FineST_tutorial_data/OrderData/position_order.csv' \
   --reduced_mtx_path 'FineST_tutorial_data/OrderData/matrix_order.npy' \
   --figure_save_path 'FineST_tutorial_data/Figures/' \
   --save_data_path 'FineST_tutorial_data/SaveData/' \
   --patch_size 112 \
   --weight_w 0.5

Key parameters:

• --dataset_class: 'Visium16' (HIPT, patch_size=64), 'Visium64' (Virchow2, patch_size=112), or 'VisiumHD'

• --image_class: 'HIPT' or 'Virchow2' (must match Step0)

• --weight_save_path: (optional) Path to pre-trained weights to skip training

Expected output:

• Average correlation of all spots: ~0.85

• Average correlation of all genes: ~0.88

Output files:

• Figures/weights[timestamp]/: Trained model weights (.pt) and logs (.log)

• Figures/Results[timestamp].log: Complete execution log

• Figures/: Visualization plots (.pdf, .svg)

• SaveData/: Processed AnnData files (adata_count.h5ad, adata_norml.h5ad, adata_infer.h5ad, etc.)

• OrderData/position_order.csv: Ordered tissue positions

• OrderData/matrix_order.npy: Ordered gene expression matrix

Option B: Visium HD

python ./demo/Step1_FineST_train_infer.py

Step2: Super-resolution spatial RNA-seq imputation (Visium)

• Step2_High_resolution_imputation.py predicts super-resolved gene expression using image segmentation (Geometric sub-spot level or Nuclei single-cell level).

• For Visium data (~5k spots, 55-um spot diameter, 100-um center-to-center distance), first interpolate additional spots between the original spots to increase resolution.

Setp2.0: Interpolate between spots

## Interpolate spots in horizontal and vertical directions
python ./demo/Spot_interpolation.py \
   --position_path FineST_tutorial_data/spatial/tissue_positions_list.csv

• Input: tissue_positions_list.csv (original within-spots)

• Output: tissue_positions_list_add.csv (interpolated between-spots, ~3x original)

Option A: sub-spot resolution

Setp A1: Extract image features for between-spots

## Option A: Using HIPT
python ./demo/Image_feature_extraction.py \
   --dataset NEW_NPC \
   --position_path FineST_tutorial_data/spatial/tissue_positions_list_add.csv  \
   --rawimage_path FineST_tutorial_data/20210809-C-AH4199551.tif \
   --scale_image False \
   --method HIPT \
   --patch_size 64 \
   --output_img FineST_tutorial_data/ImgEmbeddings/NEW_pth_64_16_image \
   --output_pth FineST_tutorial_data/ImgEmbeddings/NEW_pth_64_16 \
   --logging FineST_tutorial_data/ImgEmbeddings/Logging/ \
   --scale 0.5  # Optional, default is 0.5

## Option B: Using Virchow2
python ./demo/Image_feature_extraction.py \
   --dataset NEW_NPC \
   --position_path FineST_tutorial_data/spatial/tissue_positions_list_add.csv \
   --rawimage_path FineST_tutorial_data/20210809-C-AH4199551.tif \
   --scale_image False \
   --method Virchow2 \
   --patch_size 112 \
   --output_img FineST_tutorial_data/ImgEmbeddings/NEW_pth_112_14_image \
   --output_pth FineST_tutorial_data/ImgEmbeddings/NEW_pth_112_14 \
   --logging FineST_tutorial_data/ImgEmbeddings/
   --scale 0.5  # Optional, default is 0.5

Setp A2: Imputation at sub-spot resolution

This step supposes that the trained weight (i.e. weight_save_path in Step1) has been saved, just run the following.

## Option A: Using HIPT
python ./demo/Step2_High_resolution_imputation.py \
   --system_path '/home/lingyu/ssd/Python/FineST_submit/FineST/' \
   --parame_path 'parameter/parameters_NPC_HIPT.json' \
   --dataset_class 'Visium16' \
   --gene_selected 'CD70' \
   --LRgene_path 'FineST/datasets/LR_gene/LRgene_CellChatDB_baseline_human.csv' \
   --visium_path 'FineST_tutorial_data/spatial/tissue_positions_list.csv' \
   --imag_within_path 'FineST_tutorial_data/ImgEmbeddings/pth_64_16' \
   --imag_betwen_path 'FineST_tutorial_data/ImgEmbeddings/NEW_pth_64_16' \
   --spatial_pos_path 'FineST_tutorial_data/OrderData/position_order_all.csv' \
   --weight_save_path 'FineST_tutorial_data/Figures/weights20260204191708183236' \
   --figure_save_path 'FineST_tutorial_data/Figures/' \
   --adata_all_supr_path 'FineST_tutorial_data/SaveData/adata_imput_all_subspot.h5ad' \
   --adata_all_spot_path 'FineST_tutorial_data/SaveData/adata_imput_all_spot.h5ad'

## Option B: Using Virchow2
python ./demo/Step2_High_resolution_imputation.py \
   --system_path '/home/lingyu/ssd/Python/FineST_submit/FineST/' \
   --parame_path 'FineST_tutorial_data/parameter/parameters_NPC_virchow2.json' \
   --dataset_class 'Visium64' \
   --gene_selected 'CD70' \
   --LRgene_path 'FineST_tutorial_data/LRgene/LRgene_CellChatDB_baseline.csv' \
   --visium_path 'FineST_tutorial_data/spatial/tissue_positions_list.csv' \
   --imag_within_path 'FineST_tutorial_data/ImgEmbeddings/pth_112_14' \
   --imag_betwen_path 'FineST_tutorial_data/ImgEmbeddings/NEW_pth_112_14' \
   --spatial_pos_path 'FineST_tutorial_data/OrderData/position_order_all.csv' \
   --weight_save_path 'FineST_tutorial_data/Figures/weights20260204191708183236' \
   --figure_save_path 'FineST_tutorial_data/Figures/' \
   --adata_all_supr_path 'FineST_tutorial_data/SaveData/adata_imput_all_subspot.h5ad' \
   --adata_all_spot_path 'FineST_tutorial_data/SaveData/adata_imput_all_spot.h5ad'

Input files:

• parameters_NPC_P10125.json: The model parameters.

• LRgene_CellChatDB_baseline.csv: The genes involved in Ligand or Receptor from CellChatDB.

• tissue_positions_list.csv: It can be found in the spatial folder of 10x Visium outputs.

• AH_Patient1_pth_112_14: Image feature of within-spots from Image_feature_extraction.py.

• NEW_AH_Patient1_pth_112_14: Image feature of between-spots from Image_feature_extraction.py.

• 20240125140443830148: The trained weights from Step1.

Output files:

• patient1_adata_all.h5ad: High-resolution gene expression, at sub-spot level (16x3x resolution).

• patient1_adata_all_spot.h5ad: High-resolution gene expression, at spot level (3x resolution).

Option B: single-cell resolution

Setp B1: Nuclei segmentation (for single-cell level)

python ./demo/StarDist_nuclei_segmente.py \
   --tissue NPC_allspot_p075 \
   --out_dir FineST_tutorial_data/NucleiSegments \
   --adata_path FineST_tutorial_data/SaveData/adata_imput_all_spot.h5ad \
   --img_path FineST_tutorial_data/20210809-C-AH4199551.tif \
   --prob_thresh 0.75

• Note: Some times users may need to adjust the --prob_thresh parameter to get better segmentation results.

• The smaller the --prob_thresh, the more nuclei will be segmented, and vice versa. For NPC, the value is 0.75.

Setp B2: Extract image features for single-nuclei

## Option A: Using HIPT
python ./demo/Image_feature_extraction.py \
   --dataset sc_NPC \
   --position_path FineST_tutorial_data/NucleiSegments/NPC_allspot_p075/position_all_tissue_sc.csv  \
   --rawimage_path FineST_tutorial_data/20210809-C-AH4199551.tif \
   --scale_image False \
   --method HIPT \
   --patch_size 16 \
   --output_img FineST_tutorial_data/ImgEmbeddings/sc_pth_16_16_image \
   --output_pth FineST_tutorial_data/ImgEmbeddings/sc_pth_16_16 \
   --logging FineST_tutorial_data/ImgEmbeddings/
   --scale 0.5  # Optional, default is 0.5

## Option B: Using Virchow2
python ./demo/Image_feature_extraction.py \
   --dataset sc_NPC \
   --position_path FineST_tutorial_data/NucleiSegments/NPC_allspot_p075/position_all_tissue_sc.csv  \
   --rawimage_path FineST_tutorial_data/20210809-C-AH4199551.tif \
   --scale_image False \
   --method Virchow2 \
   --patch_size 14 \
   --output_img FineST_tutorial_data/ImgEmbeddings/sc_pth_14_14_image \
   --output_pth FineST_tutorial_data/ImgEmbeddings/sc_pth_14_14 \
   --logging FineST_tutorial_data/ImgEmbeddings/
   --scale 0.5  # Optional, default is 0.5

Setp B3: Imputation at single-cell resolution

When obtained the sc Patient1 pth 16 16 i.e., the image feature of single-nuclei from Image_feature_extraction.py, just run the following.

python ./demo/Step2_High_resolution_imputation.py \
   --system_path '/home/lingyu/ssd/Python/FineST_submit/FineST/' \
   --parame_path 'parameter/parameters_NPC_HIPT.json' \
   --dataset_class 'VisiumSC' \
   --gene_selected 'CD70' \
   --LRgene_path 'FineST/datasets/LR_gene/LRgene_CellChatDB_baseline_human.csv' \
   --image_embed_path_sc 'FineST_tutorial_data/ImgEmbeddings/sc_pth_16_16' \
   --spatial_pos_path_sc 'FineST_tutorial_data/OrderData/position_order_sc.csv' \
   --weight_save_path 'FineST_tutorial_data/Figures/weights20260204191708183236' \
   --figure_save_path 'FineST_tutorial_data/Figures/' \
   --adata_super_path_sc 'FineST_tutorial_data/SaveData/adata_imput_all_sc.h5ad'

Step3: Fine-grained LR pair and CCC pattern discovery

This step is based on SpatialDM and SparseAEH (developed by our Lab).

• SpatialDM: for significant fine-grained ligand-receptor pair selection.

• SparseAEH: for fast cell-cell communication pattern discovery, 1000 times speedup to SpatialDE.

Detailed Manual

The full manual is at FineST tutorial for installation, tutorials and examples.

Spot interpolation for Visium datasets.

• Interpolate between-spots among within-spots (Visium dataset).

Step1 and Step2 Train FineST and impute super-resolved spatial gene expression.

• On Visium (single-cell or sub-spot).

• On Visium HD (from 16um to 8um).

Step3 Fine-grained ligand-receptor (LR) pair and CCC pattern discovery.

• Nuclei-resolved ligand-receptor interaction discovery (Visium dataset).

• Super-resolved ligand-receptor interaction discovery (Visium HD dataset).

Downstream analysis Cell type deconvolution, ROI region cropping, cell-cell colocalization.

• Nuclei-resolved of Visium (use FineST-imputed data).

• Super-resolved of Visium HD (use FineST-imputed data).

• Crop region of interest (ROI) from HE image (Visium or Visium HD).

Performance evaluation of FineST vs (TESLA and iSTAR).

• PCC-SSIM-CelltypeProportion-RunTimes comparison in manuscript.

Inference comparison of FineST vs iStar (only LR genes).

• FineST on demo data.

• iStar on demo data.

Citation

If you use FineST (Accepted in principle by Nature Comm) in your research, please cite:

@misc{FineST,
   author={Li, Lingyu and Wang, Tianjie and Liang, Zhuo and Yu, Huajian and Ma, Stephanie and Yu, Lequan and Huang, Yuanhua},
   title={{FineST: Contrastive learning integrates histology and spatial transcriptomics for nuclei-resolved ligand-receptor analysis}},
   year={2026},
   note = {\url{https://github.com/StatBiomed/FineST}}
}

Contact Information

Please contact Lingyu Li ([e-mail]) or Yuanhua Huang ([e-mail]) if any enquiry.