Klaus Harter2* Abstract Novel Application of Fluorescence Lifetime and Fluorescence Microscopy Enables Quantitative Access to Subcellular Dynamics in Plant Cells Kirstin Elgass1, Katharina Caesar2, Frank Schleifenbaum1,2, York-Dieter Stierhof3, Alfred J. Meixner1*, 1 Institute for Physical and Theoretical Chemistry, University of Tu¨ bingen, Tu¨ bingen, Germany, 2 Center for Plant Molecular Biology, Department of Plant Physiology, University of Tu¨ bingen, Tu¨ bingen, Germany, 3 Center for Plant Molecular Biology, Microscopy, University of Tu¨ bingen, Tu¨ bingen, Germany 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. Citation: Elgass K, Caesar K, Schleifenbaum F, Stierhof Y-D, Meixner AJ, et al. (2009) Novel Application of Fluorescence Lifetime and Fluorescence Microscopy Enables Quantitative Access to Subcellular Dynamics in Plant Cells. PLoS ONE 4(5): e5716. doi:10.1371/journal.pone.0005716 Editor: Markus Grebe, Umea˚ Plant Science Centre, Sweden Received October 31, 2008; Accepted April 30, 2009; Published May 27, 2009 Copyright: 2009 Elgass et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. This work was funded by Ph.D. fellowships from the state Baden-Wuerttemberg to KE and KC and a DFG grant to KH (HA2146/10-1). Competing Interests: The authors have declared that no competing interests exist. * E-mail: alfred.meixner@uni-tuebingen.de (AJM); klaus.harter@zmbp.uni-tuebingen.de (KH) Introduction Fluorescence microscopy is a powerful tool for studying the subcellular partitioning and intracellular dynamics of fluorophor- tagged proteins in living cells [1–3]. It has become possible to combine fluorescence microscopy with new time-resolved laser spectroscopic methods. Higher-order pulsed laser irradiation (Stim- ulated Emission Depletion microscopy, STED) [4–5] or single molecule blinking statistics (PhotoActivated Localization Microscopy, PALM) [6] and high resolution colocalization of single molecules (STORM) [7] are used for obtaining ultra-high spatial resolution far below the diffraction limit. For observing dynamic processes associated with single molecules, techniques such as fluorescence correlation spectroscopy (FCS) are available [8–13]. However, the high resolution and sensitive fluorescence microscopy of proteins, such as membrane-bound receptor kinases, is difficult to carry out in living plant cells, especially in the context of their tissue, due to the autofluorescence of cellular compartments such as the cell wall. This problem is usually circumvented by studying proteins of higher plants in mammalian cells [14], plant protoplasts [15–16] or plant cells after plasmolysis [17]. An in vivo FCS analysis of phytochrome mobility was reported for moss protonemal tip cells [18]. However, protoplast preparation and plasmolysis induce stress responses and may modify the subcellular partitioning, intracellular dynamics and activity of the receptors. Furthermore, expression of plant receptors in heterologous systems may lead to artificial results. Due to these limitations, our knowledge on cell biological mechanisms, by which, for instance, membrane-associated receptors act at the plant cell surface, traffic inside the plant cell and induce early cellular responses, is restricted. To overcome these limitations, we applied one-chromophore fluorescence lifetime microscopy (ocFLM), combined with wave- length-selective fluorescence microscopy, to plant cells in their tissue environment in Arabidopsis thaliana at high local resolution. Our ocFLM system consisted of a confocal sample scanning microscope (CSSM), a spectrally integrating detector for measur- ing fluorescence intensity and a time-correlated single-photon counting board for recording fluorescence lifetime decays, which was custom-built in our lab [19–20]. For plant material, light- PLoS ONE | www.plosone.org 1 May 2009 | Volume 4 | Issue 5 | e5716