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Time-course imaging experiments on live organisms are critical for understanding the dynamics of growth and development. Light-sheet microscopy has advanced the field of long-term imaging of live specimens by significantly reducing photo-toxicity and allowing fast acquisition of three-dimensional data over time. However, current light-sheet technology does not allow the imaging of multiple plant specimens in parallel. To achieve higher throughput, we have developed a Multi-sample Arabidopsis Growth and Imaging Chamber (MAGIC) that provides near-physiological imaging conditions and allows high-throughput time-course imaging experiments in the ZEISS Lightsheet Z.1. Here, we illustrate MAGIC's imaging capabilities by following cell divisions, as an indicator of plant growth and development, over prolonged time periods. To automatically quantify the number of cell divisions in long-term experiments, we present a FIJI-based image processing pipeline. We demonstrate that plants imaged with our chamber undergo cell divisions for >16 times longer than those with the glass capillary system supplied by the ZEISS Z1., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Long-term grazing leads to soil degradation in Inner Mongolia grassland. Based on the Hulunbeier meadow steppe, the variation characteristics of soil nitrogen content and storage in soil layers between 0–40 cm, under six different grazing intensities, and the response of vegetation and other physical and chemical properties of soil to grazing were studied. The main results were as follows: (1) Moderate grazing increased soil total nitrogen (TN), soluble total nitrogen (STN) and microbial biomass nitrogen (MBN) contents, while heavy grazing decreased MBN content. In the year with more rain, heavy grazing increased nitrate nitrogen (NO3−-N) content and storage, while less rain increased ammonium nitrogen (NH4+-N) content. (2) The proportion of 0–40 cm nitrogen components showed an upward trend in the year with more rain, and the opposite in the years with less rainfall with the increase of grazing intensity. Soil soluble organic nitrogen (SON) and NO3−-N storages decreased and MBN storage increased in rainy years. (3) Soil nitrogen component contents and storages were correlated with plant growth status, soil moisture (SM) and soil bulk density (SBD), and were significantly negatively correlated with soil temperature (ST) and pH (p < 0.05). The content and storage of soil nitrogen were affected by grazing, soil, vegetation, meteorological and other environmental factors. Moderate grazing was more conducive to the improvement of soil nitrogen storage capacity and the healthy development of grassland. [ABSTRACT FROM AUTHOR]

This study aimed to determine the effect of tea planting age on stoichiometric ratios of microbial biomass carbon (MBC), nitrogen (MBN), and phosphorus (MBP) and soil microbial quotient (SMQ, expressed as qMBC, qMBN, and qMBP, respectively). A chronological sequence of tea plantations (3, 8, 17, 25, and 34 years) was selected in a small watershed in the Three Gorges Reservoir Area, and a slope farmland was selected as control. The results showed that with the increase of tea plantation age, soil and microbial biomass C, N, P contents, soil C: N and C: P elevated significantly, while soil N: P overall declined; the MBC: P and MBN: P increased first and then decreased, but MBC: N varied insignificantly. The tea plantation age affected SMQ notably. qMBC first decreased and then increased following the tea planting age, while qMBN and qMBP went up in a fluctuating pattern. In this study, qMBC positively correlated with soil N: P and microbial biomass C: N: P, but negatively correlated with soil C: N and C: P; on the contrary, qMBN and qMBP negatively correlated with soil N: P and microbial biomass C: N: P, but positively correlated with soil C: N and C: P. Generally, the variations of soil microbial biomass and SMQ could reflect the soil quality of tea plantations. [ABSTRACT FROM AUTHOR]

The recent significant progress in developmental bio-imaging of live multicellular organisms has been greatly facilitated by the development of light sheet fluorescence microscopy (LSFM). Both commercial and custom LSFM systems offer the best means for long-term rapid data collection over a wide field of view at single-cell resolution. This is thanks to the low light exposure required for imaging and consequent limited photodamage to the biological sample, and the development of custom holders and mounting techniques that allow for specimens to be imaged in near-normal physiological conditions. This method has been successfully applied to plant cell biology and is currently seen as one of the most efficient techniques for 3D time-lapse imaging for quantitative studies. LSFM allows one to capture and quantify dynamic processes across various levels, from plant subcellular compartments to whole cells, tissues, and entire plant organs. Here we present a method to carry out LSFM on Arabidopsis leaves expressing fluorescent markers targeted to the ER. We will focus on a protocol to mount the sample, test the phototoxicity of the LSFM system, set up a LSFM experiment, and monitor the dynamics of the ER during heat shock., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Once in a while, a field of research comes across a technological breakthrough that drives it forward. This is something that seems apparent in optical fluorescence microscopy and imaging with the advent of light sheet technology. Over a span of a few years, this technology has revolutionized the field and has brought in the concept of sheet-based plane-selective imaging with near-diffraction-limited resolution. The technology has progressed further by its integration with existing imaging methods and super-resolution techniques. Unlike existing techniques that are predominantly point-illumination-based, light sheet technology offers selectivity, single-shot interrogation, a large field-of-view, and high-speed data acquisition. Qualitatively, this has resulted in an improved signal-to-background ratio, reduced scattering, improved contrast, and the ability to interrogate a large spectrum of specimens (from cells to mammals). Recent advances have shown the reach of this emerging technology in biological sciences (developmental biology and imaging flow cytometry) and applied physics (nanolithography, beam shaping, and optical traps). The growth and adaptability of this technique are well received by the research community. The technique is expected to grow at a steady rate and has the ability to overcome the issues faced by the existing point-based techniques. In this perspective, we look at the challenges of live imaging (for mammals and plants), the methods in applied physics, and the opportunities presented by recent advances. [ABSTRACT FROM AUTHOR]

Organogenesis in plants is primarily postembryonic and relies on a strict balance between cell division and cell expansion. The root is a particularly well-suited model to study cell proliferation in detail since the two processes are spatially and temporally separated for all the different tissues. In addition, the root is amenable to detailed microscopic analysis to identify cells progressing through the cell cycle. While it is clear that cell proliferation activity is restricted to the root apical meristem (RAM), understanding cell proliferation kinetics and identifying its parameters have required much effort over many years. Here, we review the main concepts, experimental settings, and findings aimed at obtaining a detailed knowledge of how cells proliferate within the RAM. The combination of novel tools, experimental strategies, and mathematical models has contributed to our current view of cell proliferation in the RAM. We also discuss several lines of research that need to be explored in the future. [ABSTRACT FROM AUTHOR]

Background: Particle-tracking in 3D is an indispensable computational tool to extract critical information on dynamical processes from raw time-lapse imaging. This is particularly true with in vivo time-lapse fluorescence imaging in cell and developmental biology, where complex dynamics are observed at high temporal resolution. Common tracking algorithms used with time-lapse data in fluorescence microscopy typically assume a continuous signal where background, recognisable keypoints and independently moving objects of interest are permanently visible. Under these conditions, simple registration and identity management algorithms can track the objects of interest over time. In contrast, here we consider the case of transient signals and objects whose movements are constrained within a tissue, where standard algorithms fail to provide robust tracking. Results: To optimize 3D tracking in these conditions, we propose the merging of registration and tracking tasks into a registration algorithm that uses random sampling to solve the identity management problem. We describe the design and application of such an algorithm, illustrated in the domain of plant biology, and make it available as an open-source software implementation. The algorithm is tested on mitotic events in 4D data-sets obtained with light-sheet fluorescence microscopy on growing Arabidopsis thaliana roots expressing CYCB::GFP. We validate the method by comparing the algorithm performance against both surrogate data and manual tracking. Conclusion: This method fills a gap in existing tracking techniques, following mitotic events in challenging data-sets using transient fluorescent markers in unregistered images. [ABSTRACT FROM AUTHOR]

The acquisition of quantitative information on plant development across a range of temporal and spatial scales is essential to understand the mechanisms of plant growth. Recent years have shown the emergence of imaging methodologies that enable the capture and analysis of plant growth, from the dynamics of molecules within cells to the measurement of morphometricand physiological traits in field-grown plants. In some instances, these imaging methods can be parallelized across multiple samples to increase throughput. When high throughput is combined with high temporal and spatial resolution, the resulting image-derived data sets could be combined with molecular large-scale data sets to enable unprecedented systems-level computational modeling. Such image-driven functional genomics studies may be expected to appear at an accelerating rate in the near future given the early success of the foundational efforts reviewed here. We present new imaging modalities and review how they have enabled a better understanding of plant growth from the microscopic to the macroscopic scale. [ABSTRACT FROM AUTHOR]

Exposure of plants to abiotic stresses, whether individually or in combination, triggers dynamic changes to gene regulation. These responses induce distinct changes in phenotypic characteristics, enabling the plant to adapt to changing environments. For example, iron deficiency and heat stress have been shown to alter root development by reducing primary root growth and reducing cell proliferation, respectively. Currently, identifying the dynamic temporal coordination of genetic responses to combined abiotic stresses remains a bottleneck. This is, in part, due to an inability to isolate specific intervals in developmental time where differential activity in plant stress responses plays a critical role. Here, we observed that iron deficiency, in combination with temporary heat stress, suppresses the expression of iron deficiency-responsive pPYE::LUC (POPEYE::luciferase) and pBTS::LUC (BRUTUS::luciferase) reporter genes. Moreover, root growth was suppressed less under combined iron deficiency and heat stress than under either single stress condition. To further explore the interaction between pathways, we also created a computer vision pipeline to extract, analyze, and compare high-dimensional dynamic spatial and temporal cellular data in response to heat and iron deficiency stress conditions at high temporal resolution. Specifically, we used fluorescence light sheet microscopy to image Arabidopsis thaliana roots expressing CYCB1;1:GFP, a marker for cell entry into mitosis, every 20 min for 24 h exposed to either iron sufficiency, iron deficiency, heat stress, or combined iron deficiency and heat stress. Our pipeline extracted spatiotemporal metrics from these time-course data. These metrics showed that the persistency and timing of CYCB1;1:GFP signal were uniquely different under combined iron deficiency and heat stress conditions versus the single stress conditions. These metrics also indicated that the spatiotemporal characteristics of the CYCB1;1:GFP signal under combined stress were more dissimilar to the control response than the response seen under iron deficiency for the majority of the 24-h experiment. Moreover, the combined stress response was less dissimilar to the control than the response seen under heat stress. This indicated that pathways activated when the plant is exposed to both iron deficiency and heat stress affected CYCB1;1:GFP spatiotemporal function antagonistically [ABSTRACT FROM AUTHOR]

To quantitatively understand biological processes that occur over many hours or days, it is desirable to image multiple samples simultaneously and automatically process and analyze the resulting datasets. Here, we present a complete multi-sample preparation, imaging, processing, and analysis workflow to determine the development of the vascular volume in zebrafish. Up to five live embryos were mounted and imaged simultaneously over several days using selective plane illumination microscopy (SPIM). The resulting large imagery dataset of several terabytes was processed in an automated manner on a high-performance computer cluster and segmented with a novel segmentation approach that uses images of red blood cells as training data. This analysis yielded a precise quantification of growth characteristics of the whole vascular network, head vasculature, and tail vasculature over development. Our multi-sample platform demonstrates effective upgrades to conventional single-sample imaging platforms and paves the way for diverse quantitative long-term imaging studies. [ABSTRACT FROM AUTHOR]

Plants exhibit an intriguing morphological and physiological plasticity that enables them to thrive in a wide range of environments. To understand the cell biological basis of this unparalleled competence, a number ofmethodologies have been adapted or developed over the last decades that allow minimal or non-invasive live-cell imaging in the context of tissues. Combined with the ease to generate transgenic reporter lines in specific genetic backgrounds or accessions, we are witnessing a blooming in plant cell biology. However, the imaging of plant cells entails a number of specific challenges, such as high levels of autofluorescence, light scattering that is caused by cell walls and their sensitivity to environmental conditions. Quantitative live-cell imaging in plants therefore requires adapting or developing imaging techniques, as well as mounting and incubation systems, such as micro-fluidics. Here, we discuss some of these obstacles, and review a number of selected state-of-the-art techniques, such as two-photon imaging, light sheet microscopy and variable angle epifluorescence microscopy that allow high performance and minimal invasive live-cell imaging in plants. [ABSTRACT FROM AUTHOR]

Background: Characterizing the behaviors of dynamic systems requires capturing them with high temporal and spatial resolution. Owing to its transparency and genetic tractability, the Arabidopsis thaliana root lends itself well to live imaging when combined with cell and tissue-specific fluorescent reporters. We developed a novel 4D imaging method that utilizes simple confocal microscopy and readily available components to track cell divisions in the root stem cell niche and surrounding region for up to 1 week. Results: Using this method, we performed a direct measurement of cell division intervals within and around the root stem cell niche. The results reveal a short, steep gradient of cell division rates in proximal stem cells, with progressively more rapid cell division rates from quiescent center (QC), to cells in direct contact with the QC (initials), to their immediate daughters, after which division rates appear to become more homogeneous. Conclusions: These results provide a baseline to study how perturbations in signaling could affect cell division patterns in the root meristem. This new setup further allows us to finely analyze meristematic cell division rates that lead to patterning. [ABSTRACT FROM AUTHOR]