Quantifying photothrombotic ischemic volume in the intact brain using light sheet microscopy

Traditional methods for assessing photothrombotic stroke rely on in vivo imaging techniques or ex vivo histological analyses. Unlike in vivo modalities such as magnetic resonance imaging (MRI), light sheet fluorescence microscopy (LSFM) provides cellular-level high-resolution imaging without motion artifacts and can capture fine-scale morphological features of infarcts. In addition, compared with conventional histology, LSFM preserves organ integrity as the entire brain is imaged without the need for serial sectioning, thereby enabling accurate volumetric reconstruction of ischemic lesions. Here, we introduce an in-depth semi-automated method for reliable quantification of stroke volume using LSFM in optically cleared whole mouse brains following photothrombotic stroke. We demonstrate that the infarct can be delineated via endogenous autofluorescence, providing a reproducible and robust method for ischemic volume assessment. Our data show that LSFM-based stroke volume measurements are strongly correlated with in vivo laser speckle contrast imaging, in vivo MRI, and cresyl violet histology measures of stroke volume. Moreover, we show that the ischemic core remains autofluorescent regardless of the tissue preparation method, supporting the applicability of LSFM for both freshly processed and long-term stored samples. Overall, our findings validate LSFM as a reliable, versatile, and powerful alternative method for stroke volume quantification, offering significant advantages for experimental stroke research.