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Novel application of fluorescence lifetime and fluorescence microscopy enables quantitative access to subcellular dynamics in plant cells.

Elgass K, Caesar K, Schleifenbaum F, Stierhof YD, Meixner AJ, Harter K - PLoS ONE (2009)

Bottom Line: However, although established in the physical sciences, these techniques are rarely applied to cell biology in the plant sciences.We show a rapid, brassinolide-induced cell wall expansion and a fast BR-regulated change in the BRI1-GFP fluorescence lifetime in the plasmamembrane in vivo.Both cell wall expansion and changes in fluorescence lifetime reflect early BR-induced and BRI1-dependent physiological or signalling processes.

View Article: PubMed Central - PubMed

Affiliation: Institute for Physical and Theoretical Chemistry, University of Tübingen, Tübingen, Germany.

ABSTRACT

Background: Optical and spectroscopic technologies working at subcellular resolution with quantitative output are required for a deeper understanding of molecular processes and mechanisms in living cells. Such technologies are prerequisite for the realisation of predictive biology at cellular and subcellular level. However, although established in the physical sciences, these techniques are rarely applied to cell biology in the plant sciences.

Principal findings: Here, we present a combined application of one-chromophore fluorescence lifetime microscopy and wavelength-selective fluorescence microscopy to analyse the function of a GFP fusion of the Brassinosteroid Insensitive 1 Receptor (BRI1-GFP) with high spatial and temporal resolution in living Arabidopsis cells in their tissue environment. We show a rapid, brassinolide-induced cell wall expansion and a fast BR-regulated change in the BRI1-GFP fluorescence lifetime in the plasmamembrane in vivo. Both cell wall expansion and changes in fluorescence lifetime reflect early BR-induced and BRI1-dependent physiological or signalling processes. Our experiments also show the potential of one-chromophore fluorescence lifetime microscopy for the in vivo monitoring of the biochemical and biophysical subcellular environment using GFP fusion proteins as probes.

Significance: One-chromophore fluorescence lifetime microscopy, combined with wavelength-specific fluorescence microscopy, opens up new frontiers for in vivo dynamic and quantitative analysis of cellular processes at high resolution which are not addressable by pure imaging technologies or transmission electron microscopy.

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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 (50×50 µm) 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.
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pone-0005716-g001: 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 (50×50 µm) 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.

Mentions: 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 and 512 nm of BRI1-GFP expressing hypocotyl cells (Fig. 1B), the lifetime was characterized by a mono-exponential decay (Fig. 1C) typical for GFP (Figure S1) [26]–[27]. In contrast, the background autofluorescence, which was recorded at the identical wavelength range in wildtype hypocotyl cells, strongly deviated from the mono-exponential decay function (Fig. 1D). An acceptable fit to the experimental data with respect to the average lifetime and amplitude could only be achieved by a multi-exponential decay function (Fig. 1E, see Table S1 for additional information on the fitting parameters of all lifetime decay traces shown here).


Novel application of fluorescence lifetime and fluorescence microscopy enables quantitative access to subcellular dynamics in plant cells.

Elgass K, Caesar K, Schleifenbaum F, Stierhof YD, Meixner AJ, Harter K - PLoS ONE (2009)

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 (50×50 µm) 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.
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pone-0005716-g001: 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 (50×50 µm) 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.
Mentions: 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 and 512 nm of BRI1-GFP expressing hypocotyl cells (Fig. 1B), the lifetime was characterized by a mono-exponential decay (Fig. 1C) typical for GFP (Figure S1) [26]–[27]. In contrast, the background autofluorescence, which was recorded at the identical wavelength range in wildtype hypocotyl cells, strongly deviated from the mono-exponential decay function (Fig. 1D). An acceptable fit to the experimental data with respect to the average lifetime and amplitude could only be achieved by a multi-exponential decay function (Fig. 1E, see Table S1 for additional information on the fitting parameters of all lifetime decay traces shown here).

Bottom Line: However, although established in the physical sciences, these techniques are rarely applied to cell biology in the plant sciences.We show a rapid, brassinolide-induced cell wall expansion and a fast BR-regulated change in the BRI1-GFP fluorescence lifetime in the plasmamembrane in vivo.Both cell wall expansion and changes in fluorescence lifetime reflect early BR-induced and BRI1-dependent physiological or signalling processes.

View Article: PubMed Central - PubMed

Affiliation: Institute for Physical and Theoretical Chemistry, University of Tübingen, Tübingen, Germany.

ABSTRACT

Background: Optical and spectroscopic technologies working at subcellular resolution with quantitative output are required for a deeper understanding of molecular processes and mechanisms in living cells. Such technologies are prerequisite for the realisation of predictive biology at cellular and subcellular level. However, although established in the physical sciences, these techniques are rarely applied to cell biology in the plant sciences.

Principal findings: Here, we present a combined application of one-chromophore fluorescence lifetime microscopy and wavelength-selective fluorescence microscopy to analyse the function of a GFP fusion of the Brassinosteroid Insensitive 1 Receptor (BRI1-GFP) with high spatial and temporal resolution in living Arabidopsis cells in their tissue environment. We show a rapid, brassinolide-induced cell wall expansion and a fast BR-regulated change in the BRI1-GFP fluorescence lifetime in the plasmamembrane in vivo. Both cell wall expansion and changes in fluorescence lifetime reflect early BR-induced and BRI1-dependent physiological or signalling processes. Our experiments also show the potential of one-chromophore fluorescence lifetime microscopy for the in vivo monitoring of the biochemical and biophysical subcellular environment using GFP fusion proteins as probes.

Significance: One-chromophore fluorescence lifetime microscopy, combined with wavelength-specific fluorescence microscopy, opens up new frontiers for in vivo dynamic and quantitative analysis of cellular processes at high resolution which are not addressable by pure imaging technologies or transmission electron microscopy.

Show MeSH
Related in: MedlinePlus