Gadella T W, Vereb G, Hadri A E, Röhrig H, Schmidt J, John M, Schell J, Bisseling T
Department of Molecular Biology, Wageningen Agricultural University, The Netherlands.
Biophys J. 1997 May;72(5):1986-96. doi: 10.1016/S0006-3495(97)78843-X.
A novel bioactive fluorescent nodulation (Nod) factor, NodRlv-IV(BODIPY FL-C16), has been synthesized by attaching a BODIPY FL-C16 acyl chain to the primary amino group of chitotetraose deacetylated at the nonreducing terminus by recombinant NodB. The binding of the fluorescent Nod factor to root systems of Vicia sativa was investigated with fluorescence spectral imaging microscopy (FSPIM) and fluorescence ratio imaging microscopy (FRIM). Spatially resolved fluorescence spectra of living and labeled Vicia sativa root systems were measured by FSPIM. Strong autofluorescence, inherent to many plant systems when excited at 488 nm, was corrected for by utilizing the difference in fluorescence emission spectra of the autofluorescence and NodRlv-IV(BODIPY FL-C16). A methodology is presented to break down the in situ fluorescence emission spectra into spatially resolved autofluorescence and BODIPY FL fluorescence spectra. Furthermore, an FRIM method was developed for correcting autofluorescence in fluorescence micrographs for this system. After autofluorescence correction it was shown that NodRlv-IV(BODIPY FL-C16) was concentrated in the root hairs, but was also bound to other parts of the root surface.
一种新型生物活性荧光结瘤（Nod）因子NodRlv-IV（BODIPY FL-C16）已通过将BODIPY FL-C16酰基链连接到由重组NodB在非还原末端脱乙酰化的壳四糖的伯氨基上而合成。利用荧光光谱成像显微镜（FSPIM）和荧光比率成像显微镜（FRIM）研究了荧光Nod因子与紫花苜蓿根系的结合。通过FSPIM测量了活的和标记的紫花苜蓿根系的空间分辨荧光光谱。当在488nm激发时许多植物系统固有的强自发荧光，通过利用自发荧光和NodRlv-IV（BODIPY FL-C16）的荧光发射光谱的差异进行校正。提出了一种将原位荧光发射光谱分解为空间分辨的自发荧光和BODIPY FL荧光光谱的方法。此外，还开发了一种FRIM方法用于校正该系统荧光显微照片中的自发荧光。自发荧光校正后表明，NodRlv-IV（BODIPY FL-C16）集中在根毛中，但也与根表面的其他部分结合。
