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首页> 外文会议>Label-free Biomedical Imaging and Sensing 2019 >Non-invasive Spectral Analysis of Osteogenic and Adipogenic Differentiation in Adipose Derived Stem Cells using Dark-field Hyperspectral Imaging Technique
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Non-invasive Spectral Analysis of Osteogenic and Adipogenic Differentiation in Adipose Derived Stem Cells using Dark-field Hyperspectral Imaging Technique

机译：暗视野高光谱成像技术对脂肪干细胞成骨和成脂分化的非侵入性光谱分析

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Mesenchymal stem cells derived from adult adipose tissue possess the ability to differentiate into adipocytes, osteocytes,and chondrocytes which in turn can be developed into adipose tissues, cartilages, and bones. This regenerativecharacteristics has fueled the need to define improved stem-cell analysis protocol for enabling investigation of thedifferentiation process efficiently, economically, and non-invasively by start-of-the art imaging modalities. Here, wehave demonstrated hyperspectral microscopy-based label-free imaging approach to study ASCs at a single-cell level.ASCs has been stimulated to become osteocytes using the growth media containing –glycerophosphate, L-ascorbic acid2-phosphate sesquimagnesium salt hydrate, and dexamethasone. Further, ASCs were stimulated to form adipocytes usingthe growth media containing biotin, pantothenate, bovine insulin, IBMX, penicillin, rosiglitazone, and dexamethasone.In the present study, dark-field based hyperspectral Imaging (HSI) technique has been utilized to image single as well asmultiple osteoblasts and adipocytes in salt media grown on the glass substrate. The spectral response of the cells at eachpixel of the images were recorded in the visible-NIR range (400-900 nm). Response is stored in the three dimensionaldata-cube formed with two spatial dimensions and one spectral dimension. No special tagging or staining of the ASCsand derived osteoblasts, adipocytes has been done, as more likely required in traditional microscopy techniques. Incidentlight is diffracted at multiple angles and hence scattering response received after transmission is different even within thesingle cell due to sub-cellular heterogeneities present in the control and differentiating ASCs.Based on dark-field images of control and differentiated sample, we found significant structural and spectraldistinctiveness at day 14 onwards for differentiated osteoblasts and at day 6 onwards for adipocytes. Fourier filtering ofimages provides good visual inspection of structural modifications. Spectral data from the cellular surface andintracellular markers, and secreted molecules is stored to build the spectral libraries. Matrix-assisted laserdeposition/ionization (MALDI) spectrometry technique is performed on control and differentiated cells to obtain insightof sub-cellular single molecules, mineral deposits, fats, proteins, and other biological mono-constituents. In thehyperspectral images, the entire spectrum is stored within each pixel as a vector where the number of spectral bands(wavelength range) equals vector dimension and the corresponding intensity signifies the component of the individualvector. Spectral signatures from the identified lipids are then matched to the in vitro stem-cells via spectral anglemapping (SAM) algorithms. By computing angle between two pixels, remarkable spectral similarity and dissimilarity areidentified between control and differentiated stem cells. Pseudo-colored differentiating maps are produced by calibrating‘match’ threshold. Secondary validation to the HSI is provided by evaluating optical images with template-match andedge-detection algorithms as well as second-harmonic generation microscopy to investigate osteoblasts.Establishing this label-free protocol with minimum specimen preparation enables promising outcomes to overcomephototoxicity effect of traditional microscopy such as fluorescence/staining bleaching errors. The study would lead tohigh-throughput identification of patient specific derived cells for clinical use preventing mass rejection, and advance ourunderstanding of the behavior of stem cellular clusters undergoing adipogenic and osteogenic differentiation.
机译：来自成人脂肪组织的间充质干细胞具有分化为脂肪细胞，骨细胞， 软骨细胞又可以发展成脂肪组织，软骨和骨骼。这种再生 的特性促使人们需要定义改进的干细胞分析方案，以便能够研究 先进的成像方法可有效，经济且无创地完成微分过程。在这里，我们 已经证明了基于高光谱显微镜的无标记成像方法可以在单细胞水平上研究ASC。 使用含有–甘油磷酸酯，L-抗坏血酸的生长培养基刺激ASCs成为骨细胞。 2-磷酸倍半镁盐水合物和地塞米松。此外，使用以下方法刺激ASC形成脂肪细胞 含有生物素，泛酸，牛胰岛素，IBMX，青霉素，罗格列酮和地塞米松的生长培养基。 在本研究中，基于暗场的高光谱成像（HSI）技术已被用于对单幅图像以及 在玻璃基板上生长的盐培养基中有多个成骨细胞和脂肪细胞。每个细胞的光谱响应 在可见光-NIR范围（400-900 nm）中记录图像的像素。响应存储在三维中 由两个空间维和一个光谱维组成的数据多维数据集。 ASC没有特殊标记或染色 对于衍生的成骨细胞，已经完成了脂肪细胞的培养，这是传统显微镜技术所需要的。事件 光线会在多个角度发生衍射，因此即使在光波传播范围内，透射后收到的散射响应也不同 由于存在于对照和分化ASC中的亚细胞异质性，导致单细胞分化。 根据对照和分化样品的暗场图像，我们发现了明显的结构和光谱 分化成骨细胞从第14天开始具有独特性，脂肪细胞从第6天开始具有独特性。的傅立叶滤波 图像提供了对结构修改的良好视觉检查。来自细胞表面的光谱数据和 细胞内标记物和分泌的分子被存储以建立光谱库。矩阵辅助激光 在对照细胞和分化细胞上进行沉积/电离（MALDI）光谱技术以获得洞察力 亚细胞单分子，矿物质沉积，脂肪，蛋白质和其他生物单成分组成。在里面 高光谱图像，整个光谱作为矢量存储在每个像素中，其中光谱带的数量 （波长范围）等于矢量尺寸，相应的强度表示个体的成分 向量。然后，通过光谱角度将来自鉴定出的脂质的光谱特征与体外干细胞进行匹配 映射（SAM）算法。通过计算两个像素之间的角度，可以得出显着的光谱相似度和相异度 在对照干细胞和分化干细胞之间进行鉴定。伪彩色微分图通过校准产生 “匹配”阈值。通过评估模板匹配和匹配的光学图像，可以对HSI进行二次验证。 边缘检测算法以及二次谐波显微镜技术来研究成骨细胞。 建立最少样本准备的无标签方案可以克服有希望的结果 传统显微镜的光毒性效应，例如荧光/染色漂白误差。该研究将导致 高通量鉴定可用于临床的患者特异性衍生细胞，从而防止质量排斥，并促进我们 了解干细胞簇经历成脂和成骨分化的行为。

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