Subcellular Dynamics in Plants grown seedlings expressing a BRI1-GFP fusion protein were used. BRI1, a plasmalemma-bound receptor kinase for plant steroids like brassinolide (BL) [21], recycles through endosomes [22–23] and regulates many aspects of growth and differentiation, including cell expansion [24]. As shown here, our combined optical-spectroscopic application, together with a novel data analysis approach using BRI1-GFP as a model receptor, provides unprecedented insight into cells and opens up new frontiers for in vivo dynamic, quantitative analysis of molecular and subcellular processes at high local resolution. Results Differentiation of GFP and fluorescence background To differentiate between background fluorescence and the BRI1-GFP signal, we recorded a two-dimensional fluorescence intensity image over the plasmalemma-cell wall area of BRI1- GFP-expressing and wild type seedlings in root and shoot (cotyledon) cells. In contrast to the spectra of non-transformed wild type cells (Fig. 1A and B, black dots), the spectra of the BRI1- GFP-expressing cells showed a higher overall intensity and two main peaks (Fig. 1A and B, green dots). These two peaks are typical of and specific for GFP in aqueous solution (Figure S1) [25]. However, the spectra recorded from the plants differed significantly and the shoulder amplitudes and intensity ratios of the two maxima varied between root and cotyledon cells (Fig. 1A and B). These data indicate that the degree and properties of autofluorescence depend on the cell type and the autofluorescence strongly interferes with the spectrum of GFP fluorescence emission. Secondly, we recorded fluorescence lifetime decay traces from a selected probe volume with pulsed laser excitation in living plant cells. At the maximum of fluorescence emission between 500 Figure 1. Differentiation of autofluorescence and the GFP signal in BRI1-GFP-expressing and wildtype Arabidopsis cells. (A–B) Wavelength-resolved fluorescence spectra from plasmalemma-cell wall sections of BRI1-GFP-expressing cells (green) and wildtype cells (black). The spectra were recorded in root, A, or cotyledon cells, B. The areas (50650 mm) used for recording are shown in the inlets. (C) In vivo decay trace of fluorescence lifetime from BRI1-GFP expressing hypocotyl cells fitted by a mono-exponential function (black line). The lifetime decay was measured at the peak of fluorescence intensity at 500 to 512 nm. (D) In vivo decay trace of autofluorescence lifetime in the plasmalemma-cell wall area of wildtype hypocotyl cells fitted by a mono-exponential function (black line). (E) In vivo decay trace of autofluorescence lifetime in the plasmalemma-cell wall area of wildtype hypocotyl cells fitted by a multi-exponential function (black line). The lifetime decay was measured at around 500 to 512 nm. The residuals indicate the deviation between the measured and the model decay function. In a good fit the residuals are distributed symmetrically around 0. IRF, instrument response function. doi:10.1371/journal.pone.0005716.g001 PLoS ONE | www.plosone.org 2 May 2009 | Volume 4 | Issue 5 | e5716