Label-free imaging of hemoglobin degradation and hemosiderin formation in brain tissues with femtosecond pump-probe microscopy

The degradation of hemoglobin in brain tissues results in the deposition of hemosiderin, which is a major form of iron-storage protein and closely related to neurological disorders such as epilepsy. Optical detection of hemosiderin is vitally important yet challenging for the understanding of disease mechanisms, as well as improving surgical resection of brain lesions. Here, we provide the first label-free microscopy study of sensitive hemosiderin detection in both an animal model and human brain tissues. Methods: We applied spectrally and temporally resolved femtosecond pump-probe microscopy, including transient absorption (TA) and stimulated Raman scattering (SRS) techniques, to differentiate hemoglobin and hemosiderin in brain tissues. The label-free imaging results were compared with Perls' staining to evaluate our method for hemosiderin detection. Results: Significant differences between hemoglobin and hemosiderin transient spectra were discovered. While a strong ground-state bleaching feature of hemoglobin appears in the near-infrared region, hemosiderin demonstrates pure excited-state absorption dynamics, which could be explained by our proposed kinetic model. Furthermore, simultaneous imaging of hemoglobin and hemosiderin can be rapidly achieved in both an intracerebral hemorrhage (ICH) rat model and human brain surgical specimens, with perfect correlation with Perls' staining. Conclusion: Our results suggest that rapid, label-free detection of hemosiderin in brain tissues could be realized by femtosecond pump-probe microscopy. Our method holds great potential in providing a new tool for intraoperative detection of hemosiderin during brain surgeries.