David Legland

Robust incremental compensation of the light attenuation with depth in 3D fluorescence microscopy

Fluorescent signal intensities from confocal laser scanning microscopes (CLSM) suffer from several distortions inherent to the method. Namely, layers which lie deeper within the specimen are relatively dark due to absorption and scattering of both excitation and fluorescent light, photobleaching and/or other factors. Because of these effects, a quantitative analysis of images is not always possible without correction. Under certain assumptions, the decay of intensities can be estimated and used for a partial depth intensity correction. In this paper we propose an original robust incremental method for compensating the attenuation of intensity signals. Most previous correction methods are more or less empirical and based on fitting a decreasing parametric function to the section mean intensity curve computed by summing all pixel values in each section. The fitted curve is then used for the calculation of correction factors for each section and a new compensated sections series is computed. However, these methods do not perfectly correct the images. Hence, the algorithm we propose for the automatic correction of intensities relies on robust estimation, which automatically ignores pixels where measurements deviate from the decay model. It is based on techniques adopted from the computer vision literature for image motion estimation. The resulting algorithm is used to correct volumes acquired in CLSM. An implementation of such a restoration filter is discussed and examples of successful restorations are given.

Cartography of cell morphology in tomato pericarp at the fruit scale

In fleshy fruits, the variability of cell morphology at the fruit scale is largely unknown. It presents both a huge variability and a high level of organization. Better knowledge of cell morphology heterogeneity within the fruit is necessary to understand fruit development, to model fruit mechanical behaviour, or to investigate variations of physico‐chemical measurements. A generic approach is proposed to build cartographies of cell morphology at the fruit scale, which depict regions corresponding to different cell morphologies. The approach is based on: (1) sampling the whole fruit at known positions; (2) imaging and quantifying local cell morphology; (3) pooling measurements to take biological variability into account and (4) projecting results in a morphology model of the whole fruit. The result is a synthetic representation of cell morphology variations within the whole fruit. The method was applied to the characterization of cell morphology in tomato pericarp. Two different imaging scales that provided complementary descriptions were used: 3D confocal microscopy and macroscopy. The approach is generic and can be adapted to other fruits or other products.

The preprophase band of microtubules controls the robustness of division orientation in plants

Refined understanding of the preprophase band Because plant cells do not move, plant tissues are constructed according to how they place the divisions of their constituent cells. Schaefer et al. found a mutation in the model plant Arabidopsis that abolishes a visible precursor of cell division, the preprophase band. Despite loss of the band—previously thought essential to define the division plane—the general orientations of cell division planes in the roots of these plants were normal. However, individual division orientations showed more variance than normal. Thus, the preprophase band serves to focus and refine the final orientation of the nascent cell division plane. Science, this issue p. 186 A structure thought to be required for division plane selection in plants merely refines division plane position by reducing spindle rotation. Controlling cell division plane orientation is essential for morphogenesis in multicellular organisms. In plant cells, the future cortical division plane is marked before mitotic entry by the preprophase band (PPB). Here, we characterized an Arabidopsis trm (TON1 Recruiting Motif) mutant that impairs PPB formation but does not affect interphase microtubules. Unexpectedly, PPB disruption neither abolished the capacity of root cells to define a cortical division zone nor induced aberrant cell division patterns but rather caused a loss of precision in cell division orientation. Our results advocate for a reassessment of PPB function and division plane determination in plants and show that a main output of this microtubule array is to limit spindle rotations in order to increase the robustness of cell division.

Color quantification of stained maize stem section describes lignin spatial distribution within the whole stem.

This work presents a method to quantify the lignification of maize tissues by automated color image analysis of stained maize stem cross sections. Safranin and Alcian blue staining makes lignified tissues appear red, and nonlignified tissues appear blue. Lignification is assessed by the ratio of red intensity over blue intensity. A rough quantification of global lignification is computed as the surface ratio of lignified tissues. A more precise quantification is obtained by computing profiles of red/blue intensity ratio in relation to the distance to the epidermis, depicting the spatial distribution of lignified walls within the stem. Lignification profiles are analyzed through summary parameters describing the evolution of lignification in three specific regions. The distribution of lignification can be quickly assessed depending on the position and the development stage, allowing the screening of genetic variations to be envisioned.

Stereological estimation of cell wall density of DR12 tomato mutant using three-dimensional confocal imaging

BACKGROUND AND AIMS The cellular structure of fleshy fruits is of interest to study fruit shape, size, mechanical behaviour or sensory texture. The cellular structure is usually not observed in the whole fruit but, instead, in a sample of limited size and volume. It is therefore difficult to extend measurements to the whole fruit and/or to a specific genotype, or to describe the cellular structure heterogeneity within the fruit. METHODS An integrated method is presented to describe the cellular structure of the whole fruit from partial three-dimensional (3D) observations, involving the following steps: (1) fruit sampling, (2) 3D image acquisition and processing and (3) measurement and estimation of relevant 3D morphological parameters. This method was applied to characterize DR12 mutant and wild-type tomatoes (Solanum lycopersicum). KEY RESULTS The cellular structure was described using the total volume of the pericarp, the surface area of the cell walls and the ratio of cell-wall surface area to pericarp volume, referred to as the cell-wall surface density. The heterogeneity of cellular structure within the fruit was investigated by estimating variations in the cell-wall surface density with distance to the epidermis. CONCLUSIONS The DR12 mutant presents a greater pericarp volume and an increase of cell-wall surface density under the epidermis.

Statistical mapping of maize bundle intensity at the stem scale using spatial normalisation of replicated images

The cellular structure of plant tissues is a key parameter for determining their properties. While the morphology of cells can easily be described, few studies focus on the spatial distribution of different types of tissues within an organ. As plants have various shapes and sizes, the integration of several individuals for statistical analysis of tissues distribution is a difficult problem. The aim of this study is to propose a method that quantifies the average spatial organisation of vascular bundles within maize stems, by integrating information from replicated images. In order to compare observations made on stems of different sizes and shapes, a spatial normalisation strategy was used. A model of average stem contour was computed from the digitisation of several stem slab images. Point patterns obtained from individual stem slices were projected onto the average stem to normalise them. Group-wise analysis of the spatial distribution of vascular bundles was applied on normalised data through the construction of average intensity maps. A quantitative description of average bundle organisation was obtained, via a 3D model of bundle distribution within a typical maize internode. The proposed method is generic and could easily be extended to other plant organs or organisms.

Pepsin diffusion in dairy gels depends on casein concentration and microstructure

Fundamental knowledge of gastric digestion had only focused on acid diffusion from the gastric fluid, but no data are available for pepsin diffusion. Using fluorescence recovery after photobleaching technique, diffusion coefficients D of fluorescein isothiocyanate (FITC)-pepsin were measured in rennet gels across a range of casein concentrations allowing to form networks of protein aggregates with different structures. To investigate the microstructural parameters of native gels, electron microscopy image analysis were performed and qualitatively related to diffusion behavior of FITC-pepsin in these dairy gels. This study is the first report on quantification of pepsin diffusion in dairy product. Pepsin diffusion in rennet gels depends on casein concentration and microstructure. Models of polymer science can be used to assess D in dairy gel. Such data should be confronted with pepsin activity in acidic environment, and will be very useful as input parameters in mathematical models of food degradation in the human stomach.

Stereological estimation for layered structures based on slabs perpendicular to a surface.

This paper deals with the characterization of layered structures sampled with respect to a reference surface. A scheme where thick slabs are sampled perpendicular to a curved surface is considered, resulting in a non‐uniform sampling of the structure. We present an estimation procedure based on the Horvitz–Thompson principle. An approximation of the sampling probability is proposed, which depends on the local surface curvatures, on the slab dimensions and on the intensity function of slab anchors. The practical determination of local parameters is detailed for the case of a revolution surface. The procedure is applied to the estimation of surface area density of cell walls in tomato pericarp.

Histological quantification of maize stem sections from FASGA-stained images

BackgroundCrop species are of increasing interest both for cattle feeding and for bioethanol production. The degradability of the plant material largely depends on the lignification of the tissues, but it also depends on histological features such as the cellular morphology or the relative amount of each tissue fraction. There is therefore a need for high-throughput phenotyping systems that quantify the histology of plant sections.ResultsWe developed custom image processing and an analysis procedure for quantifying the histology of maize stem sections coloured with FASGA staining and digitalised with whole microscopy slide scanners. The procedure results in an automated segmentation of the input images into distinct tissue regions. The size and the fraction area of each tissue region can be quantified, as well as the average coloration within each region. The measured features can discriminate contrasted genotypes and identify changes in histology induced by environmental factors such as water deficit.ConclusionsThe simplicity and the availability of the software will facilitate the elucidation of the relationships between the chemical composition of the tissues and changes in plant histology. The tool is expected to be useful for the study of large genetic populations, and to better understand the impact of environmental factors on plant histology.

Quantitative imaging of plants: multi-scale data for better plant anatomy

This article comments on: Staedler YM, Kreisberger T, Manafzadeh S, Chartier M, Handschuh S, Pamperl S, Sontag S, Paun O, Schönenberger J. 2017. Novel computed tomography-based tools reliably quantify plant reproductive investment. Journal of Experimental Botany 69, 525–535.