Sub-cellular spectrochemical imaging of isolated human corneal cells employing synchrotron radiation-based Fourier-transform infrared microspectroscopy
Sub-cellular spectrochemical imaging of isolated human corneal cells employing synchrotron radiation-based Fourier-transform infrared microspectroscopy
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DOI：
10.1039/c2an36197c
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发表时间：
2013-01-01
期刊：
影响因子：
4.2
通讯作者：
Martin, Francis L.
中科院分区：
文献类型：
--
作者：
Fogarty, Simon W.;Patel, Imran I.;Martin, Francis L.
了解干细胞(SC)生物学仍然具有挑战性，角膜是少数几个能够很好地描述干细胞原位位置的人类组织之一。用荧光激活细胞分选技术(FACS)从活体组织中分离出单个人角膜上皮细胞，并将其划分为假定的干细胞、传递放大(TA)细胞和终末分化(TD)细胞。利用基于同步辐射的傅里叶变换红外(SR-FTIR)显微光谱和焦平面阵列(FPA)，实现了对未染色的孤立细胞的亚细胞空间分辨率分析，这是由于12束准直光束布置的亮度允许快速获取光谱。提取红外光谱，并对红外光谱进行预处理。随后的分类和从FPA图像得到的红外光谱的多变量分析被用来研究类之间的生物分子变化。从SC细胞到TA细胞再到TD细胞，细胞特有的光谱类别逐渐分离。在DNA、蛋白质和脂类光谱区识别出多个不同的吸收峰，可区分可能的干细胞、TA细胞和TD细胞。DNA区(1080和1225 cM(-1))和部分蛋白质区(1443 cM(-1))主要分离TA细胞和TD细胞，而酰胺区和脂类(1,550,1650和1740 cM(-1))分离TA细胞和TD细胞。扫描电子显微镜图像验证了不同分离细胞类型的外部表型特征。这些发现突出了SR-FTIR显微光谱学在区分干细胞、TA细胞和TD细胞方面的适用性，并表明通过传统的免疫标记方法进行细胞分类可以通过使用光谱生物标记物来极大地辅助。
Understanding stem cell (SC) biology remains challenging and one of the few human tissues within which their in situ location is well characterized is the cornea. Individual human corneal epithelial cells were isolated from biopsies of live tissues using fluorescence-activated cell sorting (FACS); these were divided into putative SCs, transit-amplifying (TA) cells and terminally-differentiated (TD) cells. Employing synchrotron radiation-based Fourier-transform infrared (SR-FTIR) microspectroscopy with a focal plane array (FPA), sub-cellular spatial resolution analysis of unstained isolated cells was achieved as a consequence of the brilliance of a 12 collimated beams arrangement allowing rapid spectral acquisition. Infrared (IR) spectra were extracted and pre-processed. Subsequent categorization with multivariate analysis of IR spectra derived from FPA images was used to investigate biomolecular changes between classes. A progressive segregation in cell-specific spectral categories with differentiation from SC to TA cell to TD cell was noted. Multiple different absorption peaks that discriminated putative SCs, TA cells and TD cells across DNA, protein and lipid spectral regions were identified. DNA regions (1080 and 1225 cm(-1)) and some protein regions (1443 cm(-1)) primarily segregated SCs from TA cells and TD cells, whilst amide regions and lipids (1,550, 1650 and 1740 cm(-1)) segregated TA cells and TD cells. Scanning electron microscopy images verified the external phenotypic characteristics of the different isolated cell types. These findings highlight the applicability of SR-FTIR microspectroscopy towards distinguishing SCs, TA cells and TD cells, and suggest that cellular classification via traditional methods of immunolabelling can be greatly aided by the use of spectral biomarkers.