21 Feb An ImageJ workflow for automated laser capture microdissection of subcellular structures

A nice collaboration between Bordeaux’ facilities has emerged from a user request, wanting to automate laser-microdissection of subcellular structures in the aim of deciphering their composition by proteomics.

When trying to combine highly demanding technics such as laser-microdissection and proteomics, one would come across two types of difficulties: 1-the required user input to delineate and isolate structures of interest; 2-quantities of material required to faithfully analyse the protein content of samples.

Unfortunately, the Zeiss PALM system we used was not provided with an API (application programming interface): a direct implementation of third-party functionalities is impossible. We circumvented this drawback by writing an ImageJ workflow, made of two parts: a plugin converting ROI Manager stored delineations into “elements” file (the input ROI format for the Zeiss device); and a toolset performing the actual detection.

The full automation is made in two steps. First the sample is scanned by the LCM device, collecting individual images of each visited field. As images are saved to the disk, the workflow will automatically process them. ROIs are extracted, and converted into the “elements” files using the plugin. In a second step, the user has to push this file to the LCM software, relax, and let the system do the job! Using this semi-automated optimized workflow, we routinely successfully were able to isolate 900 structures per hour, on average.

This proof-of-concept study was performed on invadosomes, but could well be used for any kind of detectable structures, so if you’re interested, do not hesitate to contact us (Marlène Maitre for microdissection, Frédéric Saltel for proteomics and Fabrice Cordelières for automation).

The full study is about to be published and the workflow is already available on GitHub: https://github.com/fabricecordelieres/IJ_PALM_Zeiss_workflow. On this repository, you’ll find a movie about how the workflow goes, and a poster we’ve presented during NeuBIAS2018 conference.