Raman‐based label‐free microscopic analysis of the pancreas in living zebrafish larvae

Advanced microscopy techniques, combined with a diverse set of fluorescent probes, provide valuable tools for uncovering insights into biological systems and addressing fundamental research questions. However, the need to develop and use genetic tags and probe markers presents notable challenges. Coherent Raman scattering microscopy offers a label-free alternative, enabling live-cell imaging of cellular structures without the need for labeling. Leveraging the benefits of Raman microscopy, we aim to analyze the pancreas in living zebrafish larvae and to evaluate chemical changes in pancreatic exocrine and endocrine compartments following exocrine damage. Here, we present a protocol for Raman-based label-free microscopic analysis of the pancreas in living zebrafish larvae. Using forward stimulated Raman scattering (F-SRS) and epi coherent anti-Stokes Raman scattering (E-CARS), zebrafish pancreatic structures were analyzed and validated. Vibrational Raman spectra between 450 and 3100 cm^-1 were acquired to identify chemical structural features within pancreatic regions. Raman imaging allows discrimination of distinct structures at 2850 and 2934 cm^-1 in pancreatic exocrine and endocrine regions, which could mainly correspond to lipids and proteins, respectively. Exocrine damage causes a significant reduction in both the number and size of exocrine granules. Moreover, changes at 2934 cm^-1 suggested chemical alterations in both exocrine and beta-cell regions. In conclusion, SRS and CARS provide a powerful, label-free approach for live-cell imaging and chemical analysis in islet biology. Given the relative straightforward applicability in the pancreas, we anticipate broad implementation of Raman microscopy in other organs and across various biomedical research fields.