Live monitoring of plant redox and energy physiology with genetically encoded biosensors

Genetically encoded biosensors pave the way for understanding plant redox dynamics and energy metabolism on cellular and subcellular levels.

ADVANCES

• Methodological advances in fluorescent protein-based in vivo biosensing have been instrumental for several paradigm shifts in our understanding of cell physiology, metabolism and signaling.
• An increasing number of genetically encoded biosensors has been used to dissect the dynamics of several distinct redox couples and energy physiology in plants.
• In vivo monitoring using biosensors has pioneered the simultaneous read-out of different physiological parameters in different subcellular locations by parallelized plate reader-based, multiwell fluorimetry, or expression strategies for multiple sensors in parallel.
• Sensing dynamic changes in hydrogen peroxide levels is possible with sensors of the HyPer family, or roGFP fusion variants with a thiol peroxidase.
• Peredox and SoNar family sensors enable direct visualization of NADH/NAD⁺, while iNAP family sensors respond to NADPH concentration in plants.
• Sensor variants with different sensitivity ranges enable use of the most appropriate variant for the specific in vivo environment or experimental scope.

     

mMaple: A Photoconvertible Fluorescent Protein for Use in Multiple Imaging Modalities

Recent advances in fluorescence microscopy have extended the spatial resolution to the nanometer scale. Here, we report an engineered photoconvertible fluorescent protein (pcFP) variant, designated as mMaple, that is suited for use in multiple conventional and super-resolution imaging modalities, specifically, widefield and confocal microscopy, structured illumination microscopy (SIM), and single-molecule localization microscopy. We demonstrate the versatility of mMaple by obtaining super-resolution images of protein organization in Escherichia coli and conventional fluorescence images of mammalian cells. Beneficial features of mMaple include high photostability of the green state when expressed in mammalian cells and high steady state intracellular protein concentration of functional protein when expressed in E. coli. mMaple thus enables both fast live-cell ensemble imaging and high precision single molecule localization for a single pcFP-containing construct.     

A Microfluidic Platform for Correlative Live-Cell and Super-Resolution Microscopy

Recently, super-resolution microscopy methods such as stochastic optical reconstruction microscopy (STORM) have enabled visualization of subcellular structures below the optical resolution limit. Due to the poor temporal resolution, however, these methods have mostly been used to image fixed cells or dynamic processes that evolve on slow time-scales. In particular, fast dynamic processes and their relationship to the underlying ultrastructure or nanoscale protein organization cannot be discerned. To overcome this limitation, we have recently developed a correlative and sequential imaging method that combines live-cell and super-resolution microscopy. This approach adds dynamic background to ultrastructural images providing a new dimension to the interpretation of super-resolution data. However, currently, it suffers from the need to carry out tedious steps of sample preparation manually. To alleviate this problem, we implemented a simple and versatile microfluidic platform that streamlines the sample preparation steps in between live-cell and super-resolution imaging. The platform is based on a microfluidic chip with parallel, miniaturized imaging chambers and an automated fluid-injection device, which delivers a precise amount of a specified reagent to the selected imaging chamber at a specific time within the experiment. We demonstrate that this system can be used for live-cell imaging, automated fixation, and immunostaining of adherent mammalian cells in situ followed by STORM imaging. We further demonstrate an application by correlating mitochondrial dynamics, morphology, and nanoscale mitochondrial protein distribution in live and super-resolution images.     

3D-SIM结构照明超分辨率显微镜实现蛋白质在植物亚细胞器内的定位

基因表达产物蛋白质的亚细胞定位是解析基因生物学功能的重要证据之一。近年来出现的超分辨率光学成像技术已成功应用于人类和动物细胞中,预示着显微成像技术继激光共聚焦技术后的又一重要进步。由于植物细胞的特殊性和成像技术的研发取向,超分辨率光学成像技术在植物细胞蛋白质亚细胞定位的应用尚未见报道。该研究利用Delta Vision OMX显微镜技术,克服了叶绿体基粒中叶绿素自发荧光与融合蛋白荧光不易区分的缺陷,解决了受分辨率局限无法将植物细胞中蛋白质在亚细胞器内可视化精确定位的技术难题,成功地将植物蔗糖合成酶Zm SUS-SH1定位在烟草表皮细胞叶绿体基粒周围。该研究同时建立了一套基于撕片制片法的简便OMX显微镜制片方法,并针对OMX显微成像技术在植物细胞中蛋白质亚细胞定位的应用进行了讨论。     

基于受激发射损耗显微术的活细胞和活体超分辨成像

细胞作为生命体基本的结构和功能单元，在生物、医学等领域有着非常重要的研究意义。随着现代科学和技术的发展，科学家们借助电镜对细胞以及细胞器的空间结构已经有非常清晰的认识，但是对它们的功能以及细胞之间的相互作用却了解得非常少，而这恰恰又是疾病治疗和药物开发亟需了解的信息，因此对离体活细胞（简称活细胞）和活体生物组织细胞（简称活体细胞）中亚细胞器的研究变得非常重要。然而细胞中许多细胞器的结构在纳米量级，传统的光学成像技术由于受到光学衍射极限的限制是无法观察到纳米量级的生物结构，因此光学超分辨成像技术是目前研究亚细胞器结构和功能的有效工具。在所有光学超分辨显微技术中，受激发射损耗显微术（stimulated emission depletionmicroscopy,STED）由于具有实时成像、三维超分辨和断层成像的能力，非常适合用于纳米尺度的活细胞和活体细胞成像研究，而且STED超分辨成像技术经过近几十年的发展，已经广泛用于活细胞甚至活体小鼠细胞的超分辨动态观测。本文总结了近年来活细胞和活体小鼠神经元细胞等领域STED超分辨成像的研究进展，介绍了用于活细胞和活体细胞STED超分辨成像的荧光染料...     

Zooming in on biological processes with fluorescence nanoscopy

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Highlights► Fluorescence nanoscopy can be used to decipher subcellular structures in biological systems. ► We review the underlying principles and biological application of STED, PALM, and STORM. ► For super-resolution imaging methods, there is a tradeoff between temporal and spatial resolution. ► Improving probe brightness and probe photostability are key factors for achieving higher resolution.     

Light Sheet Fluorescence Microscopy: A Review

Light sheet fluorescence microscopy (LSFM) functions as a non-destructive microtome and microscope that uses a plane of light to optically section and view tissues with subcellular resolution. This method is well suited for imaging deep within transparent tissues or within whole organisms, and because tissues are exposed to only a thin plane of light, specimen photobleaching and phototoxicity are minimized compared to wide-field fluorescence, confocal, or multiphoton microscopy. LSFMs produce well-registered serial sections that are suitable for three-dimensional reconstruction of tissue structures. Because of a lack of a commercial LSFM microscope, numerous versions of light sheet microscopes have been constructed by different investigators. This review describes development of the technology, reviews existing devices, provides details of one LSFM device, and shows examples of images and three-dimensional reconstructions of tissues that were produced by LSFM.     

Recent Advances in Super-Resolution Fluorescence Imaging and Its Applications in Biology

Fluorescence microscopy has become an essential tool for biological research because it can be minimally invasive, acquire data rapidly, and target molecules of interest with specific labeling strategies. However, the diffraction-limited spatial resolution, which is classically limited to about 200 nm in the lateral direction and about 500 nm in the axial direction, hampers its application to identify delicate details of subcellular structure. Extensive efforts have been made to break diffraction limit for obtaining high-resolution imaging of a biological specimen. Various methods capable of obtaining super-resolution images with a resolution of tens of nanometers are currently available. These super-resolution techniques can be generally divided into three primary classes： （1） patterned illumination- based super-resolution imaging, which employs spatially and temporally modulated illumination light to reconstruct sub-diffraction structures; （2） single-molecule localization-based super-resolution imaging, which localizes the profile center of each individual fluo- rophore at subdiffraction precision; （3） bleaching/blinking-based super-resolution imaging. These super-resolution techniques have been utilized in different biological fields and provide novel insights into several new aspects of life science. Given unique technical merits and commercial availability of super-resolution fluorescence microscope, increasing applications of this powerful technique in life science can be expected.     

Super-Resolution Imaging Strategies for Cell Biologists Using a Spinning Disk Microscope

In this study we use a spinning disk confocal microscope (SD) to generate super-resolution images of multiple cellular features from any plane in the cell. We obtain super-resolution images by using stochastic intensity fluctuations of biological probes, combining Photoactivation Light-Microscopy (PALM)/Stochastic Optical Reconstruction Microscopy (STORM) methodologies. We compared different image analysis algorithms for processing super-resolution data to identify the most suitable for analysis of particular cell structures. SOFI was chosen for X and Y and was able to achieve a resolution of ca. 80 nm; however higher resolution was possible >30 nm, dependant on the super-resolution image analysis algorithm used. Our method uses low laser power and fluorescent probes which are available either commercially or through the scientific community, and therefore it is gentle enough for biological imaging. Through comparative studies with structured illumination microscopy (SIM) and widefield epifluorescence imaging we identified that our methodology was advantageous for imaging cellular structures which are not immediately at the cell-substrate interface, which include the nuclear architecture and mitochondria. We have shown that it was possible to obtain two coloured images, which highlights the potential this technique has for high-content screening, imaging of multiple epitopes and live cell imaging.     

Mechanisms of resistance and virulence in parasitic plant–host interactions

Parasitic plants pose a major biotic threat to plant growth and development and lead to losses in crop productivity of billions of USD annually. By comparison with “normal” autotrophic plants, parasitic plants live a heterotrophic lifestyle and rely on water, solutes and to a greater (holoparasitic plants) or lesser extent (hemiparasitic plants) on sugars from other host plants. Most hosts are unable to detect an infestation by plant parasites or unable to fend off these parasitic invaders. However, a few hosts have evolved defense strategies to avoid infestation or protect themselves actively post-attack often leading to full or partial resistance. Here, we review the current state of our understanding of the defense strategies to plant parasitism used by host plants with emphasis on the active molecular resistance mechanisms. Furthermore, we outline the perspectives and the potential of future studies that will be indispensable to develop and breed resistant crops.

Some plants are able to recognize parasitic plants as attacking pathogens and can fend them off by inducing defense responses.

Advances

• Receptor proteins have been discovered in host plants (i.e. sunflower, tomato, or cowpea) that detect parasitic plants as an invading pathogen and further induce plant immunity and resistance responses in hosts leading to a parasite rejection.
• Molecular patterns exist in parasitic plants that can be specifically detected by host plant receptors.
• The host plant receptors require co-receptors and signaling components (i.e. BAK1, SOBIR1, etc.) also known from plant immunity against microbes.
• Parasitic plants evolved strategies to circumvent and to suppress host plant immunity, i.e. by manipulating host cells with siRNAs or proteins that act as effectors.
• Similar to the interaction of plants with microbial pathogens, elements of PTI and ETI can be both observed in plant–parasitic plant interactions.

     

The speciation and distribution characteristics of Cu in Phragmites australis (Cav.) Trin ex. Steudel

• Heavy metal allocation and the mechanism(s) of metal sequestration in different clonal organs, micro‐domains and subcellular structures has not been systematically studied for rhizomatous perennial plants. It is thus pertinent to investigate knowledge of the speciation and distribution characteristics of Cu in Phragmites australis to elucidating the mobility of metals in wetland plants after their uptake via root systems so as to facilitate development of strategies to enhance Cu tolerance.
• This study investigated the distributions of Cu in P. australis root, stem and leaf using ICP‐MS, synchrotron‐based X‐ray micro‐fluorescence and X‐ray absorption spectroscopy, then evaluated the effects of Cu on cellular structure and ultrastructure via transmission electron microscopy.
• The results indicate a clear preferential localisation of Cu in the roots as compared with the shoots (stems and leaves). The intensity of Cu in the vascular bundles was higher than that in the surrounding epidermis and the endodermis and parenchyma outside the medullary cavity. The dominant chemical form of Cu in P. australis was similar to Cu citrate.
• The results suggest that although Cu can be easily transported into the vascular tissues in roots and stems via Cu citrate, most of the metal absorbed by plants is retained in the roots because if its high binding to the cell wall, thus preventing metal translocation to aerial parts of the plants. Therefore, P. australis showed a high capacity to accumulate Cu in roots, being therefore a suitable species for phytostabilisation interventions.

     

超分辨显微成像技术在活细胞成像中的应用与发展

超分辨显微成像技术(super-resolution microscopy,SRM)可以绕过光学衍射极限对成像分辨率的限制,让以前观察不到的纳米级结构实现可视化,这一重大研究进展推动了现代生命科学和生物医学研究的进步与发展.细胞是生物体的基本组成单位,对活细胞内部的细微结构和动力学过程进行研究是掌握生命本质必不可少的途径.但由于成像原理或条件的限制,早期的SRM技术在活细胞成像应用方面受到了不同程度的限制.近几年来,随着SRM和相关技术的发展,SRM在活细胞成像研究中的应用也越来越多.本文简要介绍目前常见的几种SRM技术的基本原理和特点,并在此基础上着重阐述它们在活细胞成像应用中所取得的最新研究进展和发展方向.     

Seeing beyond the limit: A guide to choosing the right super-resolution microscopy technique

Super-resolution microscopy has become an increasingly popular and robust tool across the life sciences to study minute cellular structures and processes. However, with the increasing number of available super-resolution techniques has come an increased complexity and burden of choice in planning imaging experiments. Choosing the right super-resolution technique to answer a given biological question is vital for understanding and interpreting biological relevance. This is an often-neglected and complex task that should take into account well-defined criteria (e.g., sample type, structure size, imaging requirements). Trade-offs in different imaging capabilities are inevitable; thus, many researchers still find it challenging to select the most suitable technique that will best answer their biological question. This review aims to provide an overview and clarify the concepts underlying the most commonly available super-resolution techniques as well as guide researchers through all aspects that should be considered before opting for a given technique.     

Efficient and cost‐effective 3D cellular imaging by sub‐voxel‐resolving light‐sheet add‐on microscopy

Light‐sheet fluorescence microscopy (LSFM) allows volumetric live imaging at high‐speed and with low photo‐toxicity. Various LSFM modalities are commercially available, but their size and cost limit their access by the research community. A new method, termed sub‐voxel‐resolving (SVR) light‐sheet add‐on microscopy (SLAM), is presented to enable fast, resolution‐enhanced light‐sheet fluorescence imaging from a conventional wide‐field microscope. This method contains two components: a miniature add‐on device to regular wide‐field microscopes, which contains a horizontal laser light‐sheet illumination path to confine fluorophore excitation at the vicinity of the focal plane for optical sectioning; an off‐axis scanning strategy and a SVR algorithm that utilizes sub‐voxel spatial shifts to reconstruct the image volume that results in a twofold increase in resolution. SLAM method has been applied to observe the muscle activity change of crawling C. elegans, the heartbeat of developing zebrafish embryo, and the neural anatomy of cleared mouse brains, at high spatiotemporal resolution. It provides an efficient and cost‐effective solution to convert the vast number of in‐service microscopes for fast 3D live imaging with voxel‐super‐resolved capability.     

Integrated and correlative high-throughput and super-resolution microscopy

We have developed a method to perform microscopic temporal and spacial multi-scale experiments by imaging cellular phenotypes of interest on complementary fluorescence microscopy systems. In a low-resolution fast data acquisition screen for phenotypic cellular responses induced by small interfering RNA (siRNA), cells in spots of siRNA cell arrays showing characteristic alterations have been selected automatically by feature space analysis. These objects were imaged on a second super-resolution dSTORM microscope (direct stochastic optical reconstruction microscopy). The coordinate transfer was based on fixed cells as reference points without the use of additional fiducial markers. This procedure is suitable to combine any kind of fluorescence microscopy technique, in order to gain further insights on the observed specimen at multiple temporal or special scales.     

Joint control of plant ecological strategy by climate,regeneration mode, and ontogeny in Northeastern Chinese forests

1. Research on how plant ecological strategies (competitive, stress‐tolerant, or ruderal) vary within species may improve our understanding of plant and community responses to climate warming and also successional changes. With increasing temperature, the importance of ruderal (R) and stress tolerance (S) components is hypothesized to decrease, while the strength of the competitive (C) component should increase. Offshoots and younger plants are predicted to have greater R and smaller S components.
2. Leaf area, leaf dry matter content, and specific leaf area were measured for 1,344 forest plants belonging to 134 species in Liangshui and Fenglin Nature Reserves in Northeastern China, and C, R, and S scores calculated for each. Linear mixed effect models were used to assess how these indicators differed among study sites (n = 2), regeneration types, ontogenetic stages, and plant life forms. The two study sites have an average annual temperature difference of 0.675°C, simulating a temperature increase of 0.630°C due to climate warming.
3. Higher temperatures reduce low‐temperature stress and frost damage, which may explain the observed decrease in R and S scores; at the same time, plant competitive ability increased, as manifested by higher C scores. This effect was most pronounced for herbaceous plants, but nearly negligible as compared to the effect of regeneration type for trees and of ontogeny for woody species. Resprouting trees and younger woody plants had higher R scores and lower S scores, a sign of adaptation to high disturbance.
4. In this study, a small increase in mean annual temperature led to shifts in CSR strategy components for herbaceous species, without altering the vegetation type or community composition. Offshoots and younger plants had higher R and lower S scores, shedding light on similar changes in the ecological strategies of tree communities during secondary succession, such as the transition of Quercus mongolica coppices to forest and age‐related changes in Populus davidiana–Betula platyphylla forests.

     

Using Light Sheet Fluorescence Microscopy to Image Zebrafish Eye Development

Light sheet fluorescence microscopy (LSFM) is gaining more and more popularity as a method to image embryonic development. The main advantages of LSFM compared to confocal systems are its low phototoxicity, gentle mounting strategies, fast acquisition with high signal to noise ratio and the possibility of imaging samples from various angles (views) for long periods of time. Imaging from multiple views unleashes the full potential of LSFM, but at the same time it can create terabyte-sized datasets. Processing such datasets is the biggest challenge of using LSFM. In this protocol we outline some solutions to this problem. Until recently, LSFM was mostly performed in laboratories that had the expertise to build and operate their own light sheet microscopes. However, in the last three years several commercial implementations of LSFM became available, which are multipurpose and easy to use for any developmental biologist. This article is primarily directed to those researchers, who are not LSFM technology developers, but want to employ LSFM as a tool to answer specific developmental biology questions. Here, we use imaging of zebrafish eye development as an example to introduce the reader to LSFM technology and we demonstrate applications of LSFM across multiple spatial and temporal scales. This article describes a complete experimental protocol starting with the mounting of zebrafish embryos for LSFM. We then outline the options for imaging using the commercially available light sheet microscope. Importantly, we also explain a pipeline for subsequent registration and fusion of multiview datasets using an open source solution implemented as a Fiji plugin. While this protocol focuses on imaging the developing zebrafish eye and processing data from a particular imaging setup, most of the insights and troubleshooting suggestions presented here are of general use and the protocol can be adapted to a variety of light sheet microscopy experiments.     

Plant–plant communication and community of herbivores on tall goldenrod

1. The volatiles from damaged plants induce defense in neighboring plants. The phenomenon is called plant–plant communication, plant talk, or plant eavesdropping. Plant–plant communication has been reported to be stronger between kin plants than genetically far plants in sagebrush.
2. Why do plants distinguish volatiles from kin or genetically far plants? We hypothesize that plants respond only to important conditions; the induced defense is not free of cost for the plant. To clarify the hypothesis, we conducted experiments and investigations using goldenrod of four different genotypes.
3. The arthropod community on tall goldenrods were different among four genotypes. The response to volatiles was stronger from genetically close plants to the emitter than from genetically distant plants from the emitter. The volatiles from each genotype of goldenrods were different; and they were categorized accordingly. Moreover, the arthropod community on each genotype of goldenrods were different.
4. Synthesis: Our results support the hypothesis: Goldenrods respond to volatiles from genetically close plants because they would have similar arthropod species. These results are important clues elucidating adaptive significance of plant–plant communication.

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Applications of STED fluorescence nanoscopy in unravelling nanoscale structure and dynamics of biological systems

Fluorescence microscopy, especially confocal microscopy, has revolutionized the field of biological imaging. Breaking the optical diffraction barrier of conventional light microscopy, through the advent of super-resolution microscopy, has ushered in the potential for a second revolution through unprecedented insight into nanoscale structure and dynamics in biological systems. Stimulated emission depletion (STED) microscopy is one such super-resolution microscopy technique which provides real-time enhanced-resolution imaging capabilities. In addition, it can be easily integrated with well-established fluorescence-based techniques such as fluorescence correlation spectroscopy (FCS) in order to capture the structure of cellular membranes at the nanoscale with high temporal resolution. In this review, we discuss the theory of STED and different modalities of operation in order to achieve the best resolution. Various applications of this technique in cell imaging, especially that of neuronal cell imaging, are discussed as well as examples of application of STED imaging in unravelling structure formation on biological membranes. Finally, we have discussed examples from some of our recent studies on nanoscale structure and dynamics of lipids in model membranes, due to interaction with proteins, as revealed by combination of STED and FCS techniques.     

Nanoscale Imaging of Caveolin-1 Membrane Domains In Vivo

Light microscopy enables noninvasive imaging of fluorescent species in biological specimens, but resolution is generally limited by diffraction to ~200–250 nm. Many biological processes occur on smaller length scales, highlighting the importance of techniques that can image below the diffraction limit and provide valuable single-molecule information. In recent years, imaging techniques have been developed which can achieve resolution below the diffraction limit. Utilizing one such technique, fluorescence photoactivation localization microscopy (FPALM), we demonstrated its ability to construct super-resolution images from single molecules in a living zebrafish embryo, expanding the realm of previous super-resolution imaging to a living vertebrate organism. We imaged caveolin-1 in vivo, in living zebrafish embryos. Our results demonstrate the successful image acquisition of super-resolution images in a living vertebrate organism, opening several opportunities to answer more dynamic biological questions in vivo at the previously inaccessible nanoscale.