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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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Measurement ranges for comparison between GFP-fluorescence and cell wall-derived autofluorescence.(A) Overlap of the fluorescence spectrum of purified GFP in water (green) and the autofluorescence spectrum (blue) of wildtype Arabidopsis hypocotyl cells. The broad band of the spectrum (500–640 nm, blue) originates from the cell wall, the sharp peak at 680 nm originates from chlorophyll. The grey hatched areas show the spectral ranges used for recording GFP-fluorescence (around 500 nm) and autofluorescence, respectively (around 600 nm). (B) Fluorescence decay trace recorded in the 500 nm region (left hatched bar in A) in a BRI1-GFP expressing hypocotyl cell, fitted by a mono-exponential function (black line), proves the main presence of GFP-fluorescence. (C) Fluorescence decay trace recorded in the 600 nm region (right hatched bar in A) in a BRI1-GFP expressing hypocotyl cell, fitted by a mono-exponential function (black line), proves the dominant presence of autofluorescence. IRF, instrument response function.
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pone-0005716-g003: Measurement ranges for comparison between GFP-fluorescence and cell wall-derived autofluorescence.(A) Overlap of the fluorescence spectrum of purified GFP in water (green) and the autofluorescence spectrum (blue) of wildtype Arabidopsis hypocotyl cells. The broad band of the spectrum (500–640 nm, blue) originates from the cell wall, the sharp peak at 680 nm originates from chlorophyll. The grey hatched areas show the spectral ranges used for recording GFP-fluorescence (around 500 nm) and autofluorescence, respectively (around 600 nm). (B) Fluorescence decay trace recorded in the 500 nm region (left hatched bar in A) in a BRI1-GFP expressing hypocotyl cell, fitted by a mono-exponential function (black line), proves the main presence of GFP-fluorescence. (C) Fluorescence decay trace recorded in the 600 nm region (right hatched bar in A) in a BRI1-GFP expressing hypocotyl cell, fitted by a mono-exponential function (black line), proves the dominant presence of autofluorescence. IRF, instrument response function.

Mentions: In addition, the differences of GFP and background fluorescence with respect to their spectra and fluorescence lifetime enabled us to choose the appropriate experimental set-up for the distinction of subcellular compartments such as the plasmalemma and the cell wall (Fig. 3). Thus, we used the emission range at around 500 nm for recording GFP fluorescence and the range at around 600 nm for recording cell wall autofluorescence (hatched bars in Fig. 3A).


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)

Measurement ranges for comparison between GFP-fluorescence and cell wall-derived autofluorescence.(A) Overlap of the fluorescence spectrum of purified GFP in water (green) and the autofluorescence spectrum (blue) of wildtype Arabidopsis hypocotyl cells. The broad band of the spectrum (500–640 nm, blue) originates from the cell wall, the sharp peak at 680 nm originates from chlorophyll. The grey hatched areas show the spectral ranges used for recording GFP-fluorescence (around 500 nm) and autofluorescence, respectively (around 600 nm). (B) Fluorescence decay trace recorded in the 500 nm region (left hatched bar in A) in a BRI1-GFP expressing hypocotyl cell, fitted by a mono-exponential function (black line), proves the main presence of GFP-fluorescence. (C) Fluorescence decay trace recorded in the 600 nm region (right hatched bar in A) in a BRI1-GFP expressing hypocotyl cell, fitted by a mono-exponential function (black line), proves the dominant presence of autofluorescence. IRF, instrument response function.
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Related In: Results  -  Collection

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pone-0005716-g003: Measurement ranges for comparison between GFP-fluorescence and cell wall-derived autofluorescence.(A) Overlap of the fluorescence spectrum of purified GFP in water (green) and the autofluorescence spectrum (blue) of wildtype Arabidopsis hypocotyl cells. The broad band of the spectrum (500–640 nm, blue) originates from the cell wall, the sharp peak at 680 nm originates from chlorophyll. The grey hatched areas show the spectral ranges used for recording GFP-fluorescence (around 500 nm) and autofluorescence, respectively (around 600 nm). (B) Fluorescence decay trace recorded in the 500 nm region (left hatched bar in A) in a BRI1-GFP expressing hypocotyl cell, fitted by a mono-exponential function (black line), proves the main presence of GFP-fluorescence. (C) Fluorescence decay trace recorded in the 600 nm region (right hatched bar in A) in a BRI1-GFP expressing hypocotyl cell, fitted by a mono-exponential function (black line), proves the dominant presence of autofluorescence. IRF, instrument response function.
Mentions: In addition, the differences of GFP and background fluorescence with respect to their spectra and fluorescence lifetime enabled us to choose the appropriate experimental set-up for the distinction of subcellular compartments such as the plasmalemma and the cell wall (Fig. 3). Thus, we used the emission range at around 500 nm for recording GFP fluorescence and the range at around 600 nm for recording cell wall autofluorescence (hatched bars in Fig. 3A).

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