Microspectroscopic imaging of nodulation factor-binding sites on living vicia sativa roots using a novel bioactive fluorescent nodulation factor

Theodorus W.J. Gadella, György Vereb, Az Eddine Hadri, Horst Röhrig, Jürgen Schmidt, Michael John, Jeff Schell, Ton Bisseling

Research output: Contribution to journalArticle

22 Citations (Scopus)

Abstract

A novel bioactive fluorescent nodulation (Nod) factor, NodRIv-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 NodRIv-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 NodRIv-IV(BODIPY FL- C16) was concentrated in the root hairs, but was also bound to other parts of the root surface.

Original languageEnglish
Pages (from-to)1986-1996
Number of pages11
JournalBiophysical journal
Volume72
Issue number5
DOIs
Publication statusPublished - May 1997

ASJC Scopus subject areas

  • Biophysics

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