School of Pharmacy and Biomedical Sciences, Portsmouth University, Portsmouth, Hampshire, UK.
J Clin Pathol. 2013 Apr;66(4):312-8. doi: 10.1136/jclinpath-2012-201098. Epub 2013 Feb 7.
Fourier transform infrared (FT-IR) imaging is increasingly being applied to biomedical specimens, but strong IR absorption by water complicates live cell imaging. This study investigates the viability of adherent epithelial cells maintained for short periods under mineral oils in order to facilitate live cell spectroscopy using FT-IR with subsequent imaging.
The MGH-U1 urothelial or CaCo2 colorectal cancer cell lines were grown on plastic surfaces or mid-range infrared transparent windows. Medium in established cultures was replaced with paraffin mineral oil, or Fluorolube, for up to 2 h, and viability assessed by supravital staining. Drug handling characteristics were also assessed. Imaging of preparations was attempted by reflectance and transmission using a Varian FT-IR microscope.
Cells covered by mineral oil remained viable for 2 h, with recovery into normal medium possible. MTT ((3-(4,5-dimethylthlazol-2-yl)-2,5-diphenyl tetrazolium) conversion to crystalline formazan and differential patterns of drug uptake were maintained. The combination of a calcium fluoride substrate, Fluorolube oil, and transmission optics proved best for spectroscopy. Spectral features were used to obtain images of live cells.
The viability of cells overlaid with IR transparent oils was assessed as part of a technique to optimise conditions for FT-IR imaging. Images of untreated cells were obtained using both reflectance and transmission. This represents an effective means of imaging live cells by IR spectroscopy, and also means that imaging is not necessarily a terminal event. It also increases options for producing images based on real-time biochemistry in a range of in vitro experimental and 'optical biopsy' contexts.
傅里叶变换红外（FT-IR）成像技术越来越多地应用于生物医学标本，但水的强红外吸收使活细胞成像变得复杂。本研究旨在研究短时间内在矿物油下维持的贴壁上皮细胞的活力，以便使用 FT-IR 进行活细胞光谱学研究，并随后进行成像。
MGH-U1 尿路上皮或 CaCo2 结直肠癌细胞系在塑料表面或中红外透明窗上生长。在已建立的培养物中，用石蜡矿物油或 Fluorolube 替代培养基，最多 2 小时，并通过活体染色评估细胞活力。还评估了药物处理特性。通过使用 Varian FT-IR 显微镜进行反射和透射尝试对制剂进行成像。
覆盖矿物油的细胞在 2 小时内保持活力，并且可以恢复到正常培养基中。MTT（（3-（4,5-二甲基噻唑-2-基）-2,5-二苯基四唑）转化为结晶 formazan 以及药物摄取的差异模式得以维持。氟化钙基底、Fluorolube 油和透射光学的组合被证明最适合用于光谱学。光谱特征用于获得活细胞的图像。
作为优化 FT-IR 成像条件的技术的一部分，评估了覆盖有红外透明油的细胞的活力。使用反射和透射两种方法获得了未处理细胞的图像。这代表了通过红外光谱对活细胞进行成像的有效手段，并且也意味着成像不一定是一个终点事件。它还增加了基于一系列体外实验和“光学活检”背景下实时生物化学的图像生成选项。
