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Introduction: Research and diagnosis in medicine and biology often require the assessment of a large amount of microscopy image data. Although on the one hand, digital pathology and new bioimaging technologies find their way into clinical practice and pharmaceutical research, some general methodological issues in automated image analysis are still open. Methods: In this study, we address the problem of fitting the parameters in a microscopy image segmentation pipeline. We propose to fit the parameters of the pipeline′s modules with optimization algorithms, such as, genetic algorithms or coordinate descents, and show how visual exploration of the parameter space can help to identify sub-optimal parameter settings that need to be avoided. Results: This is of significant help in the design of our automatic parameter fitting framework, which enables us to tune the pipeline for large sets of micrographs. Conclusion: The underlying parameter spaces pose a challenge for manual as well as automated parameter optimization, as the parameter spaces can show several local performance maxima. Hence, optimization strategies that are not able to jump out of local performance maxima, like the hill climbing algorithm, often result in a local maximum.