Hyperspectral Stimulated Raman scattering (hsSRS) microscopy has recently emerged as a powerful non-destructive technique for label-free chemical imaging of biological samples. In most hsSRS imaging experiments, the SRS spectral range is limited by the total bandwidth of the excitation laser to ~300 cm-1 and spectral resolution of ~25 cm-1. Here we present a novel approach for broadband hsSRS microscopy based on parabolic fiber amplification to provide linearly chirped broadened Stokes pulses. This novel hsSRS instrument provides >600 cm-1 spectral coverage and ~10 cm-1 spectral resolution. We further demonstrated broadband hsSRS imaging of the entire Raman fingerprint region for resolving distribution of major biomolecules in fixed cells. Moreover, we applied broadband hsSRS in imaging amyloid plaques in human brain tissue with Alzheimer’s disease.
Improving the sensitivity of mammograms in breast cancer screening has increased the detection of suspicious findings such as calcifications and masses at the cost of a high false positive rate (55-85%). Additionally, the diagnostic interpretation of biopsies varies (75.3% concordance), leading to suboptimal treatments and poor patient outcomes. The goal of this pilot study is to investigate whether the chemical composition of breast calcifications, present in more than 80% of mammograms, can be used to improve breast lesion classification. We hypothesized that the spatial and compositional variation of breast calcifications strongly correlates with breast malignancy. To test this hypothesis, we used an advanced Raman imaging technique called hyperspectral stimulated Raman scattering (hsSRS) microscopy to study 12 patient cases (30 calcifications). We characterized unique Raman signatures of type I (calcium oxalate) and type II (calcium hydroxyapatite) calcifications in archival breast tissue at high speed and spatial resolution with hsSRS microscopy. We found that the carbonation level of hydroxyapatite decreases when comparing benign and atypical ductal hyperplasia. However, the average carbonation of hydroxyapatite was highly variable in fibroadenoma cases (3±0.6%) and DCIS (4±1.1%). In the case of DCIS, the carbonation of hydroxyapatite varied relative to the grade and the neoplastic microenvironment (nearby inflammation, necrosis, and more.) In high-grade DCIS, the carbonation was lowest around the periphery where the contact with neoplastic cells was present. Our preliminary results indicate that microcalcifications change with the neoplastic microenvironment. Further studies of neoplastic progression in association with microcalcifications can improve the statistical value of the correlation.
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