In this step, you need to upload reference and alignment images for processing.
Set ONE reference image
Supported formats: TIFF, image TAR.GZ, or .stereo files.
TIFF
image TAR.GZ
.stereo
You can click to replace the reference image.
Add alignment images
Supported formats: TIFF, image TAR.GZ, or .stereo files.
TIFF
image TAR.GZ
.stereo
If you want to add more alignment images, please click to add images.
Note:
The reference image decides SN.
You can remove the specified image by closing the corresponding tab.
Configure Image Information
Input file format
Stainting type: ssDNA/DAPI/H&E
Stainting type: IF
TIFF
Fill-in manually
Fill-in manually
.stereo/compressed Image TAR.GZ
Auto-detection based on the file metadata
Auto-detection based on the file metadata.
If you have uploaded the DAPI image in the reference field, you can close the repeated DAPI image tab in the alignment field.
Step 2: Adjust orientation
Use the rotate or flip tools to roughly align the image with the reference.
If you cannot locate common features between reference and alignment images, please contact the person of sample preparation.
Locate image features
Rotate
Flip
You can change orientations with these icons:
Rotate 90°
Flip horizontal
Flip vertical
To reset all changes, click "Reset".
Please confirm that each image to be aligned is roughly in the same orientation as the reference image, without mirroring or 90-degree rotation.
Example of mismatch orientation
Click "Next" to confirm the changes.
Step 3: Mark key points
Mark points on corresponding features in the reference and alignment images.
Stage 1: Enable sync zoom (First 3 pairs)
Zoom into the cell-level, and click 3 pairs of corresponding features.
After marking 3 valid pairs, the software automatically triggers synchronization. Zoom/pan adjustments made in the reference image will now sync across all alignment images.
Stage 2: Complete point marking
Continue marking 3 additional pairs of features to activate "Next" button.
Add more pairs if needed: If you have more items to align and want to ensure a thorough job, you can create additional pairs. There is no upper limit, so feel free to add as many as you need. Ideally, 25~30 points. Please click "Next" to confirm the changes.
Tricks in DAPI+mIF alignment
Due to properties in DAPI+mIF staining, locating features based on cell morphology is not an easy task. If your DAPI+ mIF imaging is on the same chip, you can identify the chip outline by tuning brightness, normalization, and contrast.
Key Functionalities
Function
Description
Example
Undo
Safely remove incorrect or redundant points by reversing the order of addition.
Clear all points
All marked points will be deleted.
Draft recovery
If you skip clicking 'Next' in Step 3 before proceeding to another step, your current progress will not be saved.
When returning later, you can click 'Recover' to retrieve the unsaved draft.
Step 4: Check & Refine
Check the alignment result first. If anything looks off, fine-tune with move, scale, or rotate. If there are multiple images, review and adjust each one separately.
View reference image
View alignment image
Change alignment image
Adjust Transformations
Function
Example
Step move
Scale
Rotation
Adjust Visual properties for improved overlay visibility. Note: these settings will not be saved when exporting.
Saturation for colored images
Opacity
Brightness
Contrast
Normalize
Pseudo-colors for grayscale images
You can check the transformation values of alignment images.
Step5: Export
Export aligned images in TIFF or RPI format for analysis or visualization.
Export in TIFF format
Naming rules: <SN>_<stain_type>.tif
If you input .stereo or compressed image TAR.GZ files, and they have been registered with matrices, the software will flip them.
to replace the reference image.
to add images.
Rotate 90°
Flip horizontal
Flip vertical
brightness, 
normalization, and 
contrast.
Step move
Scale
Rotation
