Correlative Imaging of Fluorescent Proteins in Resin-Embedded Plant Material¹

Abstract

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 mm 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.

In situ structure of FtsZ mini-rings in Arabidopsis chloroplasts

“…Both acrylic resins used, Lowicryl HM20 and LR White, proved capable of maintaining the fluorescent protein signal. This is in agreement with previous reports where methacrylates, LR White or Lowicryl have been employed in conjunction with plant and animal tissue and yeast cells [45][46][47][48].…”

Chemical reactivation of quenched fluorescent protein molecules enables resin-embedded fluorescence microimaging

“…Although quenching still exists, fluorescence signals have been successfully detected and analysed in resin-embedded cultured cells and small tissues for correlative microscopy studies78910. However, these methods are difficult to transplant to process thick tissue blocks.…”

Standard fluorescent proteins as dual-modality probes for correlative experiments in an integrated light and electron microscope

“…Different CLEM workflows have been developed for different applications [5,6]. Recently, a number of inresin fluorescence (IRF) methods have been reported that preserve the activity of GFP and other fluorophores through sample processing for electron microscopy (EM), thereby improving the accuracy of FP localisation, and promoting standard fluorophores to the role of dual-modality probes [7][8][9][10][11]. Generally, cells and tissues expressing FPs are immobilised using high-pressure freezing [12], after which the water in the sample is substituted at low temperature for a solvent containing a small percentage of uranyl acetate and water, before infiltration with and polymerisation in a water-tolerant resin.…”