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A year ago, in September 2022, we were contacted via the Euro-BioImaging infrastructure by a user from the Max Planck Institute of Psychiatry in Germany who was working on the analysis of mouse stress and social avoidance behavior (Fig. 1). Mouse brain hemispheres were labeled against c-Fos, an immediate early gene, and cleared using iDISCO. They were subsequently imaged using Light Sheet microscope Ultramicroscope II from MIltenyi Biotech and stitched with Big Stitcher. The goal was to identify c-Fos in number and intensity between groups compared to control in different brain regions. This is enabled by annotating brain images into the Allen Brain Atlas, a platform for visualizing single cell data across the mammalian brain and identifying brain regions.

An already published analysis pipeline included ClearMap, a toolbox for the analysis and registration of 3D datasets obtained via light sheet microscopy from cleared tissues. ClearMap is written in Python, so it can be easily accessed through, for example, a Jupyter Notebook. The first part of the analysis included segmentation of the c-Fos foci, elastic tranformation of the Allen Brain Atlas images to match the autofluorescence channel of the acquired brain images and final identification of the brain regions. The annotation to the atlas was followed by statitical analysis in MATLAB using a custom written script. In addition to comparing cell numbers and fluorescence intensities in different brain regions, cell density maps were constructed (Fig 2.).

The project required some time to set up - terms, data transfer, format of results or timeline had to be agreed upon. Its implementation started in February 2023 and lasted for 8 months with gradual refinement of the requirements and the form of the output. In total, around 1TB of data was transferred and processed, and the final results, both image and statistical outputs, were sent to the end user. Such external projects are of great importance for microscopy facilities, as they bring new opportunities and the acquired experience can be further used in solving internal tasks.

If you require assistance with your image data analysis and processing tasks, don’t hesitate to contact us or visit our facility.

VMCF, in collaboration with bioMT, Department of Biochemistry and Cell Biology, Dartmouth College, Hanover NH, USA, chaired the BioImage Analysis in Super-resolution Microscopy workshop. The workshop included introductory lectures on superresolution microscopy techniques, hands-on exercises using Fiji and ThunderSTORM software, and discussions on software tools such as Noise2Void, StarDist, Huygens, Ilastik, and CLIJ2. There was also a scientific talk on cardiomyocytes and a session on customizing Fiji with ImageJ Macro. The VMCF staff members were represented by Zuzana Burdíková, Ph.D., Martin Schätz, Ph.D. and Jakub Soukup, Ph.D. The workshop was developed in collaboration with Zdenek Švindrych, Ph.D., and Pat Robinson, Ph.D. from bioMT.

The combination of the expertise of the experienced lecturers contributed to the success of the workshop, both from VMCF (CR) and bioMT (USA). The workshop was supported by COBRE P20-GM113132 (NIH) and LM2018129 Czech-BioImaging grant. The feedback from the students who attended the workshop was overwhelmingly positive. Their feedback indicated that the workshop was very valuable and provided them with a deeper understanding of Super-resolution Microscopy and image analysis. The participants especially appreciated the hands-on nature of the program and the expertise of faculty, including those from VMCF and bioMT, in helping participants gain practical skills in image analysis and high-resolution microscopy.