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

Abstract

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.

Keywords: hemoglobin; hemosiderin; pump-probe microscopy; stimulated Raman scattering; transient absorption.

Figure 1

Schematics of the experimental design. (A) Optical transitions of TA and SRS processes. (B) Optical layout of the pump-probe microscope. (C) Linear absorption spectra of hemoglobin and hemosiderin. EOM: electro-optical modulator; DM: dichroic mirror; SP: short pass filter; PD: photodiode; LIA: lock-in amplifier.

Figure 2

Characteristic transient optical responses of hemoglobin and hemosiderin. TA dynamics and images of hemoglobin (A) and hemosiderin (B), respectively. (C) TA images of a mixture of the two compounds at different probe wavelengths. Scale bar: 20 µm.

Figure 3

Spectrally and temporally resolved TA spectra of hemoglobin (A-B) and hemosiderin (C-D). Fitted curves are drawn as solid lines. (E) TA spectra taken at different time delays. (F) Extracted A₃ component from equation (1) at various probe wavelengths.

Figure 4

Imaging RBCs and hemosiderin in rat brain tissue at 802 nm probe wavelength. (A-C) Pump-probe images taken at different time delays. (D) Composite image showing the distribution of hemoglobin (red), hemosiderin (cyan) and brain tissues (green), using linear recombination of the data in (A-C). (E) TA curves of hemoglobin, hemosiderin and lipid. Lipid response reflects the cross-correlation between femtosecond pump and probe pulse. Scale bar: 20 µm.

Figure 5

Validation of pump-probe microscopy in brain tissues of a rat ICH model. (A) Bright-field images of the whole fresh section and thin frozen section of an ICH rat brain. (B) TA/SRS image of a typical coronal brain section, mapping hemoglobin (red), hemosiderin (cyan) and brain tissues (green). (C) Zoomed-in region of (B) with the contrast of hemoglobin and hemosiderin only. (D) Perls' staining of the same tissue section in (C). Scale bar: 500 µm.

Figure 6

Hemosiderin detection in human CCM tissues. (A) TA/SRS image and (B) Perls' staining of the same CCM paraffin section. (C) MRI scan of a left frontal CCM with a hypo-intense ring (red arrow) on T₂ sequence, resulted from hemosiderin deposition. (D) Illustration of differences in hemosiderin deposition of CCM lesion. Gross margins may still contain hemosiderin (brown dots). (E) TA/SRS image of the resected fresh CCM lesion margin, showing the vessel lumen-like space in the brain tissue. (F) Resected tissue farther away from the lesion with much fewer hemosiderin deposits. Cyan: hemosiderin; gray: brain tissues. Scale bar: 30 µm.