Abstract
Thin tissue slice based histology has been used as a gold standard for disease diagnosis since over a hundred years ago. However, histopathological evaluation on two-dimensional slides suffers from large variations due to limited sampling. To improve the diagnostic accuracy, three-dimensional (3D) histology is performed through serial sectioning, staining, imaging and reconstruction of individual slices, which is highly time-consuming and labor intensive. We developed a volumetric stimulated Raman scattering (SRS) imaging method, which provides histology-like information in 3D context without the need for staining with dyes. Using a small molecule clearing agent, formamide, we performed tissue clearing within 30 min and achieved an imaging depth up to 500 µm in highly scattered tissues, including brain, kidney, liver and lung. Through a two-color SRS imaging scheme, we obtained histology-like images in cleared brain tissue slices. Our method has the potential for 3D tissue histopathology to improve the accuracy of histopathological examination.
Highlights
Pathological examination has been a gold standard for disease diagnosis and prognosis
Conventional pathology is mostly performed on ultra-thin (4 to 6 μm) tissue slices stained with dyes, such as haematoxylin and eosin (H&E), to enhance structural and intracellular contrast
Stimulated Raman scattering (SRS) histopathology avoids the need for staining with dyes by detecting intrinsic chemical bond vibrations
Summary
Pathological examination has been a gold standard for disease diagnosis and prognosis. Limited sampling from a bulk of tissues increases the inter- and intra-observer variability [1], reducing the reproducibility and accuracy of two-dimensional (2D) pathology Considering these inherent drawbacks of 2D pathology, there has been an increasing demand for 3D histopathology [2]. One straightforward way to obtain 3D pathology is to perform serialsectioning, staining, imaging and reconstruction This method is inevitably time-consuming, labor-intensive and destructive to the samples, and is not widely adopted in clinical use. Substantial efforts have been devoted to developing infrared (IR) spectroscopic imaging toward chemical histopathology in the past decade [5,6,7,8] This method suffers from relatively low resolution (>2 μm) and limited penetration depth (∼30 μm). Attenuated total reflection IR improves the resolution down to submicron scale, it is only feasible for surface imaging (penetration depth 1-3 μm) [9]
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