ADVANCING TWO-PHOTON CALCIUM IMAGING IN GLIOMA RESEARCH THROUGH OPEN-HARDWARE AND OPEN-SOFTWARE INNOVATIONS

Glioblastoma multiforme (GBM) is the most aggressive form of brain cancer, with a median survival of approximately 15 months. Current standard treatments (surgical resection, chemotherapy, electrotherapy, and anti-epileptic medication) have not produced substantial improvements in clinical outcomes. While several structural and molecular aspects of this interaction have been described, few studies have investigated how the functional activity of peritumoral brain tissue is altered.
We set out to characterize functional changes in peritumoral cortical circuits using in vivo two-photon calcium imaging in mouse visual cortex, since local computations, intracortical connectivity, and cell-type–specific responses to visual stimuli are well characterized in the healthy brain. Developing this paradigm requires us to overcome two major methodological challenges.
Firstly, functional two-photon measurements must be performed during visual stimulation, which introduces significant stray-light contamination. To solve this, we developed the Photon Shield, an open-hardware light-shielding system consisting of a 3D-printed headplate and a rail-mounted objective shield that provides robust and reproducible protection against stimulation-induced stray light, enabling artefact-free imaging.
Secondly, accurate interpretation of neural activity requires software tools for validating the output of widely used calcium-signal analysis algorithms, particularly CaImAn’s CNMF and OnACID pipelines. For this purpose, we developed Pluvianus, a stand-alone graphical user interface for visual exploration and quality control of CaImAn results. Pluvianus facilitates transparent curation, and improves the reliability of extracted neural signals.
Together, light-artefact suppression with the Photon Shield and robust signal-quality assessment with Pluvianus represent important steps toward accurately characterizing functional alterations in GL261-implanted Thy1-GCaMP6s mouse peritumoral cortical circuits.