Deep Ultraviolet Mapping of Intracellular Protein and Nucleic Acid in Femtograms per Pixel
Deep Ultraviolet Mapping of Intracellular Protein and Nucleic Acid in Femtograms per Pixel
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DOI：
10.1002/cyto.a.21111
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发表时间：
2011-11-01
期刊：
影响因子：
3.7
通讯作者：
Ehrlich, Daniel J.
中科院分区：
文献类型：
--
作者：
Cheung, Man C.;Evans, James G.;Ehrlich, Daniel J.
通过使用成像分光光度法在200- 280 nm波长范围内的配对图像，我们直接映射细胞内的核酸和蛋白质分布在整个人口的中国仓鼠卵巢（CHO-K1）细胞。宽带1003物镜的数值孔径为1.2 NA（甘油浸泡）和一种新型的激光诱导等离子体点源产生高对比度的图像与短（类似于100毫秒）曝光和横向分辨率接近200 nm，很容易解决内部细胞器。在420个CHO-K1细胞和477个细胞核的群体中，我们发现G1全细胞核酸峰为26.6 pg，细胞核分离的总核酸峰为11.4 pg，并且如通过RNA酶处理推断的，G1总DNA质量为7.4 pg。在G1峰处，我们发现全细胞蛋白质质量为95.6 pg，和39.3pg的核分离蛋白质质量。发现感测肽键（220-nm）吸光度的蛋白质定量算法具有更高的信噪比，并且当与更经典的280/260-nm相比时，提供更可靠的核酸和蛋白质测定。nm算法时，用于细胞内质量映射。使用同时成像与常见的核染色剂（Hoechst 33342，Syto-14，和Sytox橙子），我们已经比较了染色模式，深紫外图像的浓缩染色质，并确认这些常见的核染色剂的偏见有关的核包装。该方法允许绝对质量测量，无需特殊的样品制备或染色。它可以与普通荧光显微镜结合使用，并对显微镜进行相对适度的修改。(C)2011年国际细胞计数促进学会
By using imaging spectrophotometry with paired images in the 200- to 280-nm wavelength range, we have directly mapped intracellular nucleic acid and protein distributions across a population of Chinese hamster ovary (CHO-K1) cells. A broadband 1003 objective with a numerical aperture of 1.2 NA (glycerin immersion) and a novel laser-induced-plasma point source generated high-contrast images with short (similar to 100 ms) exposures and a lateral resolution nearing 200 nm that easily resolves internal organelles. In a population of 420 CHO-K1 cells and 477 nuclei, we found a G1 whole-cell nucleic acid peak at 26.6 pg, a nuclear-isolated total nucleic acid peak at 11.4 pg, and, as inferred by RNase treatment, a G1 total DNA mass of 7.4 pg. At the G1 peak, we found a whole-cell protein mass of 95.6 pg, and a nuclear-isolated protein mass of 39.3 pg. An algorithm for protein quantification that senses peptide-bond (220-nm) absorbance was found to have a higher signal-to-noise ratio and to provide more reliable nucleic acid and protein determinations when compared to more classical 280/260-nm algorithms when used for intracellular mass mapping. Using simultaneous imaging with common nuclear stains (Hoechst 33342, Syto-14, and Sytox Orange), we have compared staining patterns to deep-UV images of condensed chromatin and have confirmed bias of these common nuclear stains related to nuclear packaging. The approach allows absolute mass measurements with no special sample preparation or staining. It can be used in conjunction with normal fluorescence microscopy and with relatively modest modification of the microscope. (C) 2011 International Society for Advancement of Cytometry