Correlative Imaging of Fluorescent Proteins in Resin-Embedded Plant Material1

Fluorescent proteins (FPs) were developed for live-cell imaging and have revolutionized cell biology. However, not all plant tissues are accessible to live imaging using confocal microscopy, necessitating alternative approaches for protein localization. An example is the phloem, a tissue embedded deep within plant organs and sensitive to damage. To facilitate accurate localization of FPs within recalcitrant tissues, we developed a simple method for retaining FPs after resin embedding. This method is based on low-temperature fixation and dehydration, followed by embedding in London Resin White, and avoids the need for cryosections. We show that a palette of FPs can be localized in plant tissues while retaining good structural cell preservation, and that the polymerized block face can be counterstained with cell wall probes. Using this method we have been able to image green fluorescent protein-labeled plasmodesmata to a depth of more than 40 μm beneath the resin surface. Using correlative light and electron microscopy of the phloem, we were able to locate the same FP-labeled sieve elements in semithin and ultrathin sections. Sections were amenable to antibody labeling, and allowed a combination of confocal and superresolution imaging (three-dimensional-structured illumination microscopy) on the same cells. These correlative imaging methods should find several uses in plant cell biology.The localization of fluorescent proteins (FPs) in cells and tissues has become one of the major tools in cell biology (Tsien, 1998; Shaner et al., 2005). Advances in confocal microscopy have meant that many proteins can be tagged with appropriate fluorescent markers and tracked as they move within and between cells (Chapman et al., 2005). Additional approaches involving photobleaching and photoactivation of FPs have opened up new avenues for exploring protein dynamics and turnover within cells (Lippincott-Schwartz et al., 2003). However, not all cells are amenable to live-cell imaging, which in plants is usually restricted to surface cells such as the leaf epidermis. An example is the phloem. The delicate nature of sieve elements and companion cells, which are under substantial hydrostatic pressure, has made studies of the fine structure of these cells particularly difficult (Knoblauch and van Bel, 1998). Despite this, significant advances have been made in imaging the phloem through inventive use of imaging protocols that allow living sieve elements to be observed as they translocate assimilates (for review, see Knoblauch and Oparka, 2012). However, determining the precise localization of the plethora of proteins located within the sieve element (SE)-companion cell (CC) complex remains a technical challenge. The phloem is the conduit for long-distance movement of macromolecules in plants, including viral genomes. For several viruses, the entry into the SE-CC complex is a crucial step that determines the capacity for long-distance movement. Identifying the cell types within the phloem that restrict the movement of some viruses is technically challenging due to the small size of phloem cells and their location deep within plant organs (Nelson and van Bel, 1998).The problems associated with imaging proteins in phloem tissues prompted us to explore methods for retaining the fluorescence of tagged proteins within tissues not normally amenable to confocal imaging. Previously, we used superresolution imaging techniques on fixed phloem tissues sectioned on a Vibroslice, providing information on the association between a viral movement protein (MP) and plasmodesmata (PD) within the SE-CC complex (Fitzgibbon et al., 2010). However, we wished to explore the same cells using correlative light and electron microscopy (CLEM), necessitating the development of methods that would allow sequential imaging of cells using fluorescence microscopy and transmission electron microscopy (TEM). To this end, we developed a protocol that retains fluorescent proteins through aldehyde fixation and resin embedding.In the last 10 years there has been significant interest in imaging fluorescent proteins in semithin sections (for review, see Cortese et al., 2009). Luby-Phelps and colleagues (2003) first described a method for retaining GFP fluorescence after fixation and resin embedding, but their method has not seen widespread application. The advent of superresolution imaging techniques (for review, see Bell and Oparka, 2011) has stimulated considerable interest in this field as the retention of fluorescence in thin sections means that cells can be imaged using techniques such as photoactivation light microscopy and stochastic optical reconstruction microscopy, allowing a lateral resolution of less than 10 nm to be achieved (Subach et al., 2009; Xu et al., 2012). A number of studies have described CLEM on the same cells (Luby-Phelps et al., 2003; Betzig et al., 2006; Watanabe et al., 2011). Advances in this field were reviewed recently (Jahn et al., 2012; see contributions in Muller-Reichert and Verkade, 2012). For example, Pfeiffer et al. (2003) were able to image SEs and CCs using high-pressure freezing, followed by freeze substitution in acetone and resin embedding. They then used thick optical sections of the tissue to locate cells of interest, and these were subsequently imaged using TEM. However, there have been few attempts to retain FPs in resin-embedded plant tissues. Thompson and Wolniak (2008) described the retention of mCitrine fused to an SE-plasma membrane protein in glycol methacrylate sections. The fluorescent signal was stable using wide-field microscopy but bleached rapidly under the confocal microscope.To date, cryosections have been the preferred choice for CLEM in mammalian tissues (Watanabe et al., 2011). Recently, Lee et al. (2011) chemically fixed Arabidopsis (Arabidopsis thaliana) seedlings, cut 50-μm sections, and examined these with a confocal microscope. After confocal mapping the sections were embedded in resin and thin sectioned. These authors were able to locate the same PD pit fields using confocal and TEM, providing important information on the localization of a novel PD protein. As general rule, cryosectioning is a time-consuming process, and subcellular details may be obscured in cryosections because of poor tissue contrast (Watanabe et al., 2011). A major problem with imaging FPs in resin sections has been that GFP and its derivatives are quenched by the acidic, oxidizing conditions required for fixation, dehydration, and embedding of delicate specimens (Tsien, 1998; Keene et al., 2008). Recently, however, Watanabe et al. (2011) explored the retention of FPs in Caenorhabditis elegans cells after fixation by different aldehydes and embedding media. These authors tested a range of resins and found that Citrine and tandem dimer Eos (tdEos) could be retained in methacrylate plastic sections. This material was difficult to cut thinly (<70 nm) compared to epoxy-based resins, but the authors obtained valuable correlative images using stimulated emission depletion microscopy and photoactivation light microscopy followed by low-voltage scanning electron microscopy.Because the retention of fluorescent proteins may differ between plant and animal cells, we explored a number of approaches for retaining fluorescent proteins in resin. Using low-temperature conditions (<8°C) during fixation and dehydration, we could retain strong fluorescence prior to tissue embedding. We also explored different embedding media and found that tissue could be effectively polymerized in London Resin (LR) White while retaining sufficient fluorescence for confocal imaging. Using water-dipping lenses, we were able to detect fluorescent proteins in optical sections up to 40 μm below the surface of the block face. Ultrathin sections from the same blocks showed good structural preservation and allowed CLEM. Subsequently, we cut 1- to 2-μm sections and examined these using confocal microscopy and three-dimensional-structured illumination microscopy (3D-SIM). Sections could be counterstained with a number of conventional fluorophores and antibodies, allowing colocalization studies. These simple methods allow successive imaging of FPs with the light and electron microscope, combining the strengths of both imaging platforms. We believe this approach will have significant utility for tissues that are recalcitrant to conventional confocal imaging.