Laboratory for Functional Optical Imaging, Department of Biomedical Engineering, Columbia University, New York, New York, United States of America.
PLoS One. 2011;6(5):e19925. doi: 10.1371/journal.pone.0019925. Epub 2011 May 16.
Living tissues contain a range of intrinsic fluorophores and sources of second harmonic generation which provide contrast that can be exploited for fresh tissue imaging. Microscopic imaging of fresh tissue samples can circumvent the cost and time associated with conventional histology. Further, intrinsic contrast can provide rich information about a tissue's composition, structure and function, and opens the potential for in-vivo imaging without the need for contrast agents.
METHODOLOGY/PRINCIPAL FINDINGS: In this study, we used hyperspectral two-photon microscopy to explore the characteristics of both normal and diseased gastrointestinal (GI) tissues, relying only on their endogenous fluorescence and second harmonic generation to provide contrast. We obtained hyperspectral data at subcellular resolution by acquiring images over a range of two-photon excitation wavelengths, and found excitation spectral signatures of specific tissue types based on our ability to clearly visualize morphology. We present the two-photon excitation spectral properties of four major tissue types that are present throughout the GI tract: epithelium, lamina propria, collagen, and lymphatic tissue. Using these four excitation signatures as basis spectra, linear unmixing strategies were applied to hyperspectral data sets of both normal and neoplastic tissue acquired in the colon and small intestine. Our results show that hyperspectral unmixing with excitation spectra allows segmentation, showing promise for blind identification of tissue types within a field of view, analogous to specific staining in conventional histology. The intrinsic spectral signatures of these tissue types provide information relating to their biochemical composition.
CONCLUSIONS/SIGNIFICANCE: These results suggest hyperspectral two-photon microscopy could provide an alternative to conventional histology either for in-situ imaging, or intraoperative 'instant histology' of fresh tissue biopsies.
活体组织包含一系列固有荧光团和二次谐波产生源，这些荧光团和二次谐波产生源提供的对比度可用于对新鲜组织进行成像。对新鲜组织样本进行显微镜成像可以避免传统组织学相关的成本和时间。此外，固有对比度可以提供有关组织成分、结构和功能的丰富信息，并为无需造影剂的体内成像开辟了可能性。
方法/主要发现：在这项研究中，我们仅依赖于内源性荧光和二次谐波产生来提供对比度，使用高光谱双光子显微镜来探索正常和患病胃肠道（GI）组织的特征。通过在一系列双光子激发波长范围内获取图像，我们以亚细胞分辨率获得高光谱数据，并根据我们能够清晰地可视化形态的能力，找到特定组织类型的激发光谱特征。我们展示了存在于整个胃肠道中的四种主要组织类型的双光子激发光谱特性：上皮、固有层、胶原和淋巴组织。使用这四个激发特征作为基础光谱，我们将线性解混策略应用于从小肠和结肠获得的正常和肿瘤组织的高光谱数据集。我们的结果表明，使用激发光谱的高光谱解混可以进行分割，有望实现对视野内组织类型的盲识别，类似于传统组织学中的特定染色。这些组织类型的固有光谱特征提供了与其生化成分相关的信息。
结论/意义：这些结果表明，高光谱双光子显微镜可以替代传统组织学，用于原位成像，或者对新鲜组织活检进行术中“即时组织学”。
