Department of Electrical and Computer Engineering, 14743University of Houston, Houston, TX, USA.
Department of Hematopathology, 4002The University of Texas MD Anderson Cancer Center, Houston, TX, USA.
Appl Spectrosc. 2022 Apr;76(4):508-518. doi: 10.1177/00037028211063513. Epub 2022 Mar 2.
Collagen quantity and integrity play an important role in understanding diseases such as myelofibrosis (MF). Label-free mid-infrared spectroscopic imaging (MIRSI) has the potential to quantify collagen while minimizing the subjective variance observed with conventional histopathology. Infrared (IR) spectroscopy with polarization sensitivity provides chemical information while also estimating tissue dichroism. This can potentially aid MF grading by revealing the structure and orientation of collagen fibers. Simultaneous measurement of collagen structure and biochemical properties can translate clinically into improved diagnosis and enhance our understanding of disease progression. In this paper, we present the first report of polarization-dependent spectroscopic variations in collagen from human bone marrow samples. We build on prior work with animal models and extend it to human clinical biopsies with a practical method for high-resolution chemical and structural imaging of bone marrow on clinical glass slides. This is done using a new polarization-sensitive photothermal mid-infrared spectroscopic imaging scheme that enables sample and source independent polarization control. This technology provides 0.5 µm spatial resolution, enabling the identification of thin (≈1 µm) collagen fibers that were not separable using Fourier Transform Infrared (FT-IR) imaging in the fingerprint region at diffraction-limited resolution ( ≈ 5 µm). Finally, we propose quantitative metrics to identify fiber orientation from discrete band images (amide I and amide II) measured under three polarizations. Previous studies have used a pair of orthogonal polarization measurements, which is insufficient for clinical samples since human bone biopsies contain collagen fibers with multiple orientations. Here, we address this challenge and demonstrate that three polarization measurements are necessary to resolve orientation ambiguity in clinical bone marrow samples. This is also the first study to demonstrate the ability to spectroscopically identify thin collagen fibers (≈1 µm diameter) and their orientations, which is critical for accurate grading of human bone marrow fibrosis.
胶原数量和完整性在理解骨髓纤维化（MF）等疾病中起着重要作用。无标记中红外光谱成像（MIRSI）有可能在最小化传统组织病理学观察到的主观差异的同时对胶原进行定量。具有偏振敏感性的红外（IR）光谱提供了化学信息，同时还估计了组织二色性。这有可能通过揭示胶原纤维的结构和取向来辅助 MF 分级。同时测量胶原结构和生化特性可以在临床上转化为改善诊断，并增强我们对疾病进展的理解。在本文中，我们首次报道了来自人骨髓样本的胶原的偏振相关光谱变化。我们在之前的动物模型工作的基础上进行了扩展，并将其扩展到具有实用方法的人类临床活检中，该方法可对临床载玻片上的骨髓进行高分辨率的化学和结构成像。这是使用新的偏振敏感光热中红外光谱成像方案完成的，该方案可实现对样本和光源的独立偏振控制。该技术提供了 0.5 µm 的空间分辨率，能够识别薄（≈1 µm）的胶原纤维，这些纤维在指纹区域以衍射极限分辨率（≈5 µm）使用傅里叶变换红外（FT-IR）成像无法分离。最后，我们提出了定量指标，以从在三个偏振下测量的离散带图像（酰胺 I 和酰胺 II）中识别纤维取向。先前的研究使用了一对正交偏振测量，这对于临床样本来说是不够的，因为人体骨活检中包含具有多种取向的胶原纤维。在这里，我们解决了这一挑战，并证明了三个偏振测量对于解决临床骨髓样本中的取向模糊性是必要的。这也是首次证明能够通过光谱学识别薄的胶原纤维（≈1 µm 直径）及其取向的研究，这对于准确分级人类骨髓纤维化至关重要。
