Fluorescence in situ hybridization (FISH) analysis software is used to process and analyze FISH images to quantify and interpret the spatial distribution of nucleic acids within cells. There are several free software options available for FISH image analysis, each with its own set of features, advantages, and limitations. Below are some of the commonly used free FISH analysis software tools along with their pros and cons:

1. ImageJ/FIJI

Pros:

Open-source and free.

Highly customizable with a vast array of plugins.

User-friendly interface for basic image processing.

Widely used in the scientific community.

Cons:

Can be complex for advanced FISH analysis without the right plugins.

Steeper learning curve for new users.

May require programming skills for extensive customization.

2. CellProfiler

Pros:

Open-source and free.

Highly flexible and can be tailored to specific FISH analysis needs.

Batch processing capabilities for high-throughput analysis.

Strong community support.

Cons:

Can be time-consuming to set up for complex analyses.

The learning curve can be steep for users not familiar with image analysis pipelines.

3. QuPath

Pros:

Open-source and free.

Designed for pathology image analysis, which can include FISH.

User-friendly interface with object classification and annotation tools.

Supports both brightfield and fluorescence imaging.

Cons:

May not be as specialized for FISH as other software.

Less commonly used for FISH analysis compared to ImageJ/FIJI or CellProfiler.

4. Icy

Pros:

Open-source and free.

Offers a variety of plugins for FISH analysis.

Interactive interface for real-time analysis.

Cons:

Less well-known and therefore has a smaller user community.

The documentation and support may be less comprehensive compared to other tools.

5. Biotools

Pros:

Free for academic use.

Specialized for FISH and other cytogenetic analyses.

User-friendly with a focus on ease of use.

Cons:

May not be as flexible or extensible as open-source software.

Limited to academic use.

As for their use in impact articles, these software tools have been used in various high-impact publications. The choice of software often depends on the specific requirements of the study, the expertise of the researchers, and the resources available.

When using these tools for research that will be published in high-impact journals, it is important to ensure that the software is fit for the purpose, that the analysis is rigorous, and that the methodology is well-documented. Peer reviewers often look for the validity and reliability of the image analysis methods used in a study.