Denaturing of single electrospun fibrinogen fibers studied by deep ultraviolet fluorescence microscopy.

Denaturing of single electrospun fibrinogen fibers studied by deep ultraviolet fluorescence microscopy.
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DOI:
10.1002/jemt.20896
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发表时间:
2011-03
影响因子:
2.5
通讯作者:
Guthold, Martin
Guthold, Martin
中科院分区:
工程技术3区
文献类型:
--
作者:
Kim, Jeongyong;Song, Hugeun;Park, Inho;Carlisle, Christine R.;Bonin, Keith;Guthold, Martin

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深紫外光(DUV)显微镜是一种荧光显微镜技术,通过一些氨基酸的天然荧光对未标记蛋白质进行成像。通过对倒置光学显微镜的改造,构建了一台可激发280 nm波长的DUV荧光显微镜。此外,我们将纳米操纵器控制的微管集成到该仪器中,以精确地将皮升数量的液体输送到样本的选定区域。在原理验证实验中,我们使用该仪器原位研究了变性剂对单一的、未标记的、电纺纤维蛋白原纳米纤维的自体荧光强度的影响。纳米纤维的自发荧光发射在280 nm处被激发,在~350 nm处被检测到。一种变性剂溶液被离散地应用于纳米纤维的小的、选定的部分,并且观察到明显的局部自发荧光强度降低。这种减少归因于纤维的溶解和纤维中蛋白质的展开。
Deep ultraviolet (DUV) microscopy is a fluorescence microscopy technique to image unlabeled proteins via the native fluorescence of some of their amino acids. We constructed a DUV fluorescence microscope, capable of 280 nm wavelength excitation by modifying an inverted optical microscope. Moreover, we integrated a nanomanipulator-controlled micropipette into this instrument for precise delivery of picoliter amounts of fluid to selected regions of the sample. In proof-of-principle experiments, we used this instrument to study, in situ, the effect of a denaturing agent on the autofluorescence intensity of single, unlabeled, electrospun fibrinogen nanofibers. Autofluorescence emission from the nanofibers was excited at 280 nm and detected at ~350 nm. A denaturant solution was discretely applied to small, select sections of the nanofibers and a clear local reduction in autofluorescence intensity was observed. This reduction is attributed to the dissolution of the fibers and the unfolding of proteins in the fibers.
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