Challenges in Brillouin microscopy: Addressing heterogeneity and contamination for reliable biomechanical mapping of fresh brain tumors

Objective and RationaleBrain biomechanics is a rapidly evolving field, with mechanical properties influencing both normal development and pathological conditions such as cancer. Brillouin microscopy, a non-contact optical technique, offers a promising approach for studying the biomechanics of fresh brain tumors and organoids at subcellular resolution. However, challenges such as tissue heterogeneity and signal attenuation necessitate an in-depth evaluation of measurement strategies and potential confounding factors. MethodsFresh human brain tumor samples and tumor organoids were analyzed using Brillouin microscopy with 780 nm excitation. Measurements in the form of maps of various size were performed, and the impact of focal position, tissue heterogeneity and blood contamination on Brillouin data was assessed. Complementary Raman spectroscopy was performed as reference for tissue composition. ResultsBrillouin signal intensity decreased exponentially with depth, with valid measurements achievable up to 80 {micro}m. Low signal intensities at greater depths compromised data reliability due to fitting algorithm limitations. Structural heterogeneity, including different cell types, differentially affected signal attenuation. Blood contamination was identified as a major confounder, leading to erroneous biomechanical readings. Brillouin intensity maps provided essential quality control for accurate data interpretation. Raman spectroscopy identified the presence of blood and tissue-specific biochemical signatures, reinforcing the importance of multimodal analysis. ConclusionsBrillouin microscopy can effectively probe biomechanical properties of fresh brain tumors but is influenced by tissue heterogeneity and contaminants. Proper sample preparation, strategic focal positioning, and complementary techniques like Raman spectroscopy are critical for ensuring reliable data. These findings contribute to refining Brillouin microscopy protocols for neuro-oncological research and potential future clinical applications.