MicroSAM Usage

Step-by-step Protocol for Using MicroSAM

Micro-SAM Usage in Napari:

Once Micro-SAM is installed, follow these steps to segment your datasets:

1. Organize your data: 

• If your dataset contains multiple timepoints, these must be processed separately because Micro-SAM does not sup1port timepoints directly. 

• Organize your dataset so that each timepoint is stored as an independent TIFF file containing all Z slices. 

• If your data is originally saved as T-Z stacks, you must first split them into individual TIFF files for each timepoint (for example using ImageJ/Fiji).

2. Launch Micro-SAM from command line: 

• Open the command prompt and type the following commands to activate Micro-SAM: 

conda activate micro-sam

napari

3. Access the Micro-SAM Plugin in Napari: 

• Within Napari, navigate to: 

           Plugins > SegmentAnything for Microscopy > Annotator 3D

4. Import your image stack: 

• Load the TIFF file corresponding to one timepoint. 

           File > Open File(s)

• Select the TIFF file containing the Z-stack for a single timepoint.

5. Open and Preprocess your stack: 

• Specify the model to use and select the directory where embeddings will be saved by clicking Select Directory.

6. Compute Embeddings: 

• Click on Compute Embeddings. 

• A new folder will be created containing the embeddings for the processed timepoint.

7. Automatic Segmentation: 

• Click on the Automatic Segmentation button to generate the initial segmentations. 

• You can then adjust the parameters to optimize the segmentation results. 

8. Manual Segmentation Correction: 

• After automatic segmentation, you may need to manually correct errors. 

• To delete unwanted surfaces, use the syringe tool to click on the background and then select the surface to remove. 

• To create new cells, use prompts (e.g., rectangles or points), click on the cell of interest, and then apply changes across all Z slices by clicking Segment All Slices. 

9. Exporting Images: 

To export the image with labels in image format follow: 

• Select File > Save Selected Layers > Choose the following format “napari builtins labels (*.tif *.tiff *.bsdf. *im .*lsm .*npz .*pbm .*pcx .*pgm .*ppm .*stk)”. 

10. (Optionally) Combine Segmented Time Points: 

• After segmenting all frames, combine the segmented time points into a single folder. 

• Import this folder into ImageJ to combine the time points into a single stack.