Image analysis in fluorescence microscopy: Bacterial dynamics as a case study

The advancement of high-content optical microscopy has enabled the acquisition of very large three-dimensional (3D) image datasets. The analysis of these image volumes requires more computational resources than a biologist may have access to in typical desktop or laptop computers. This is especially true if machine learning tools are being used for image analysis. With the increased amount of data analysis and computational complexity, there is a need for a more accessible, easy-to-use, and efficient network-based 3D image processing system. The distributed and networked analysis of volumetric image data (DINAVID) system was developed to enable remote analysis of 3D microscopy images for biologists. We present an overview of the DINAVID system and compare it to other tools currently available for microscopy image analysis. DINAVID is designed using open-source tools and has two main sub-systems, a computational system for 3D microscopy image processing and analysis and a 3D visualization system. DINAVID is a network-based system with a simple web interface that allows biologists to upload 3D volumes for analysis and visualization. DINAVID enables the image access model of a center hosting image volumes and remote users analyzing those volumes, without the need for remote users to manage any computational resources. The DINAVID system, designed and developed using open-source tools, enables biologists to analyze and visualize 3D microscopy volumes remotely without the need to manage computational resources. DINAVID also provides several image analysis tools, including pre-processing and several segmentation models.

The Society of Biomolecular Imaging and Informatics First Annual Conference

Quantitative image analysis is an important tool in understanding cell fate processes through the study of cell morphological changes in terms of size, shape, number, and orientation. In this context, this work explores systematically the main challenges involved in the quantitative analysis of fluorescence microscopy images and also proposes a new protocol while comparing its outcome with the widely used ImageJ analysis. It is important to mention that fluorescence microscopy is by far most widely used in biocompatibility analysis (observing cell fate changes) of implantable biomaterials. In this study, we employed two different image analyses toolsets: (a) the conventionally employed ImageJ software, and (b) a recently developed automated digital image analyses framework, called ImageMKS. While ImageJ offers a powerful toolset for image analyses, it requires sophisticated user expertise to design and iteratively refine the analyses workflow. This workflow primarily comprises a sequence of image transformations that typically involve de-noising and labeling of features. On the other hand, ImageMKS automates the image analyses protocol to a large extent, and thereby mitigates the influence of the user bias on the final results. This aspect is addressed using a case study of C2C12 mouse myoblast cells grown on poly(vinylidene difluoride) (PVDF) based polymeric substrates. In particular, we used a number of fluorescence microscopy images of these mouse myoblasts grown on PVDF-based nanobiocomposites under the influence of electric field. In addition to the MKS workflows requiring much less user time because of their automation, it was observed that ImageMKS workflows consistently produced more reliable results that correlated better with the previously reported experimental studies.

ImageJ is a versatile and powerful tool for quantitative image analysis in microscopy. It is open-source software, platform-independent and enables students and researchers to obtain an easy but thorough introduction into image analysis. Especially the image processing package Fiji is a valuable and powerful extension of ImageJ. Several plugins and macros for single-molecule localization microscopy (SMLM) have been developed during the last decade. These novel tools cover the steps from single-molecule localization and image reconstruction to SMLM data postprocessing such as density analysis, image registration or resolution estimation. This article describes how ImageJ/Fiji can be used for image analysis, reviews existing extensions for SMLM, and aims at introducing and motivating novices and advanced SMLM users alike to explore the possibilities of ImageJ/Fiji for automated and quantitative data analysis.

The scale and complexity of images collected in biological microscopy have grown enormously over the past 30 years. The development and commercialization of multiphoton microscopy has promoted a renaissance of intravital microscopy, providing a window into cell biology in vivo. New methods of optical sectioning and tissue clearing now enable biologists to characterize entire organs at subcellular resolution. New methods of multiplexed imaging support simultaneous localization of forty or more probes at a time. Exploiting these exciting new techniques has increasingly required biomedical researchers to master procedures of image analysis that were once the specialized province of imaging experts. A primary goal of the Indiana O’Brien Center has been to develop robust and accessible image analysis tools for biomedical researchers. Here we describe biomedical image analysis software developed by the Indiana O’Brien Center over the past 25 years.

Autophagy is a conserved degradative process that is crucial for cellular homeostasis and cellular quality control via the selective removal of subcellular structures such as mitochondria. We demonstrate that a regulatory link exists between mitochondrial function and autophagy in Saccharomyces cerevisiae. During amino-acid starvation, the autophagic response consists of two independent regulatory arms-autophagy gene induction and autophagic flux-and our analysis indicates that mitochondrial respiratory deficiency severely compromises both. We show that the evolutionarily conserved protein kinases Atg1, target of rapamycin kinase complex I, and protein kinase A (PKA) regulate autophagic flux, whereas autophagy gene induction depends solely on PKA. Within this regulatory network, mitochondrial respiratory deficiency suppresses autophagic flux, autophagy gene induction, and recruitment of the Atg1-Atg13 kinase complex to the pre-autophagosomal structure by stimulating PKA activity. Our findings indicate an interrelation of two common risk factors-mitochondrial dysfunction and autophagy inhibition-for ageing, cancerogenesis, and neurodegeneration.

Automated determination of poplar chip size distribution based on combined image and multivariate analyses

Membrane Glycoproteins. I. Enzymatic Synthesis of Mannosyl Phosphoryl Polyisoprenol and Its Role as a Mannosyl Donor in Glycoprotein Synthesis (Waechter, C. J., Lucas, J. J., and Lennarz, W. J. (1973) J. Biol. Chem. 248, 7570–7579) William Joseph Lennarz was born in New York City in 1934. He received his B.S. in chemistry from Pennsylvania State University in 1956 and his Ph.D. in organic chemistry from the University of Illinois in 1959. Subsequently, he carried out postdoctoral research on fatty acid biosynthesis at Harvard University with Journal of Biological Chemistry (JBC) Classic author Konrad Bloch (1.JBC Classics: Bloch, K., and Rittenberg, D. (1942) J. Biol. Chem. 145, 625–636; Rittenberg, D., and Bloch, K. (1945) J. Biol. Chem. 160, 417–424; Bloch, K. (1945) J. Biol. Chem. 157, 661–666 (http://www.jbc.org/cgi/content/full/280/10/e7)Google Scholar). In 1962 Lennarz moved to Baltimore where he was appointed Assistant Professor in the Department of Physiological Chemistry at the Johns Hopkins School of Medicine. He was later promoted to Associate Professor of Biochemistry in 1966 and Professor in 1971. The focus of Lennarz's work at Johns Hopkins was lipids and bacterial cell surfaces. He found that mannosyl phosphoryl undecaprenol participates in the biosynthesis of mannolipids in Micrococcus lysodeikticus (2.Lahav M. Chiu T.H. Lennarz W.J. Studies on the biosynthesis of mannan in Micrococcus lysodeikticus. II. The enzymatic synthesis of mannosyl-1-phosphoryl-undecaprenol.J. Biol. Chem. 1969; 244: 5890-5898Abstract Full Text PDF PubMed Google Scholar). This work showed that lipid-linked sugars were biosynthetic precursors of envelope-associated bacterial polysaccharides. Curious as to whether polyisoprenol phosphates also served as glycosyl carriers in mammalian tissue, Lennarz and his colleagues looked at the enzymatic transfer of mannose from GDP-mannose to endogenous acceptors in cell-free preparations of bovine thyroid and hen oviduct. This is the subject of the JBC Classic reprinted here. Lennarz found that the preparations did catalyze the transfer of mannose to several endogenous acceptors including mannosyl phosphoryl polyisoprenol. Two other receptors were discovered and named “soluble mannosylated endogenous acceptor” (mannosyl s-acceptor) and “residual mannosylated endogenous acceptor” (mannosyl r-acceptor). Lennarz and his colleagues then showed that mannosyl phosphoryl polyisoprenol serves as the mannosyl donor for the synthesis of both mannosyl s- and r-acceptor. He further postulated that mannosyl s-acceptor mediates the transfer of mannosyl residues from mannosyl phosphoryl polyisoprenol to glycoproteins. From these results Lennarz concludes, “it seems possible that in eukaryotic systems, as in bacterial systems, activated lipid-linked sugars mediate the synthesis of glycose-containing macro-molecules that are associated with the membranous components of the cell.” Lennarz left Baltimore for Texas in 1983 when he was appointed Robert A. Welch Professor and Chairman of the Department of Biochemistry and Molecular Biology at the University of Texas Cancer Center, M. D. Anderson Hospital in Houston. In 1989, he joined the faculty of the State University of New York at Stony Brook and became Distinguished Professor and Chairman of the Department of Biochemistry and Cell Biology, a title he still holds today. In 1990 he founded and became Director of the Institute for Cell and Developmental Biology at Stony Brook. Lennarz continues to focus on glycoproteins, and his more recent efforts have been on the structure, biosynthesis, and function of cell surface glycoproteins and the role of cell surface proteins in fertilization and embryonic development in the sea urchin and frog. Currently, he is studying the steps involved in glycoprotein synthesis, including N-glycosylation and protein folding, as well as the functions of the glycan chains. Lennarz served as president of the American Society for Biochemistry and Molecular Biology in 1989 and was also president of both the Biochemistry Chairman's Organization and the Society for Glycobiology. He was awarded the Society for Glycobiology's Karl Meyer Award in 2004. Lennarz was a member of the Executive Committee of the International Union of Biochemistry and Molecular Biology for almost a decade. He served as co-editor-in-chief for the Encyclopedia of Biological Chemistry and was a member of the editorial board for Biochemical and Biophysical Research Communications. He was elected to the National Academy of Sciences in 1989.

Received: 2018-06-07 | Accepted: 2018-06-19 | Available online: 2018-06-30 https://doi.org/10.15414/afz.2018.21.02.77-83 The aim of this study was to distinguishing between kernels of maize hybrids by the use of image analysis tools. We analyzed 10 registered Zea mays L. hybrids (5 – dent, 2 – semi-flint to flint, and 3 – semi-flint to dent type). Different parameters on ventral, dorsal, corolla side, and lateral side cross section of kernel were measured. Sample per each hybrid comprised 50 maize kernels. Acquired bio-images were processed by software Zeiss AxioVision Rel. 4.8. We analyzed the segmented regions of interest on the kernels. The data for area (mm 2 ), height and width (mm) were gathered from these regions. The hybrid ZE EDOX significantly differed (p < 0.05) from all other hybrids almost in all traits. It is the hybrid with the smallest area of the whole kernel, floury endosperm proportion, and depressed part on corolla. The new trait the area of the depressed part on the kernel corolla was measured. The hybrids with smaller proportion of floury endosperm had smaller area of depressed part, and vice versa. The image analysis methods can usefully contribute to selection of proper hybrids for different types of use. Keywords: maize, Zea mays L., kernel, image analysis References BLASCHKE, T. et al. (2014) Geographic Object-Based Image Analysis – Towards a new paradigm. In ISPRS Journal of Photogrammetry and Remote Sensing, vol. 87, pp. 180–191. https://doi.org/10.1016/j.isprsjprs.2013.09.014 DE CARVALHO, M. L. M., VAN AELST, A. C., VAN ECK, J. W., HOEKSTRA, F. A. (1999) Pre-harvest stress cracks in maize (Zea mays L.) kernels as characterized by visual, X-ray and low temperature scanning electron microscopical analysis: effect on kernel quality. In Seed Science Research, vol. 9, pp. 227–236. DELL´AQUILA, A. (2006) Computerised seed imaging: a new tool to evaluate germination quality. In Communications in Biometry and Crop Science, vol. 1, no. 1, pp. 20–31. ERASMUS, C. (2003) Maize kernel translucency measurement by Image Analysis and its relationship to vitreousness and dry milling performance. PhD thesis. Pretoria: University of Pretoria. FOX, G. and MANLEY, M. (2009) Hardness methods for testing maize kernels. In Journal of Agricultural and Food Chemistry, vol. 57, no. 13, pp. 5647–5657. doi: https://doi.org/10.1021/jf900623w GLASBEY, C. A. and HORGAN, G. W. (2001) Image analysis in agriculture research. In Quantitative Approaches in System Analysis, special issue, vol. 23, pp. 43–54. GUELPA, A., DU PLESIS, A., KIDD, M., MANLEY, M. (2015) Nondestructive estimation of maize (Zea mays L.) kernel hardness by means of an X-ray micro-computed tomography (μCT) density calibration. In Food and Bioprocess Technology, vol. 8, no. 6, pp. 1419–1429. JANDA, J. and MICHALEC, V. (1982) Mayze. Bratislava: Priroda. 408 p. MUTTERER, J. and ZINCK, E. (2013) Quick‐and‐clean article figures with FigureJ. In Journal of Microscopy, vol. 252, pp. 89– 91. doi: https://doi.org/10.1111/jmi.12069 ROBUTTI, J. L., HOSENEY, R. C. and WASSOM, C. E. (1974). Modified opaque-2 corn endosperms. ll. Structure viewed with a scanning electron microscope. In American Association of Cereal Chemists, vol. 51, pp. 173–180. RODRIGUEZ-PULIDO, F. J. et al. (2012) Ripeness estimation of grape berries and seeds by image analysis. In Computers and Electronics in Agriculture, vol. 82, pp. 128–133. SCHINDELIN, J., RUEDEN, C. T., HINER, M. C., ELICEIRI, K. W. (2015) The ImageJ ecosystem: An open platform for biomedical image analysis. In Mol. Reprod. Dev., vol. 82, pp. 518–529. doi: https://doi.org/10.1002/mrd.22489 UCHIDA, S. (2013) Image processing and recognition for biological images. In Development, growth and differentiation, vol. 55, pp. 523–549. doi: https://doi.org/10.1111/dgd.12054 WATSON, S. A. (1987) Structure and composition. In WATSON, S. A. and RAMSTAD, P. E. (Eds.). Corn chemistry and technology. St. Paul: American Association of Cereal Chemists. WIWART M. et al. (2012) Identification of hybrids of spelt and wheat and their parental forms using shape and color descriptors. In Computers and Electronics in Agriculture, vol. 83, pp. 68–76. https://doi.org/10.1016/j.compag.2012.01.015

Over the past decade, apoptosis has emerged as an important field of study central to ongoing research in many diverse fields, from developmental biology to cancer research. Apoptosis proceeds by a highly coordinated series of events that includes enzyme activation, DNA fragmentation, and alterations in plasma membrane permeability. The detection of each of these phenotypic changes is accessible to advanced undergraduate cell and molecular biology students. We describe a 4-week laboratory sequence that integrates cell culture, fluorescence microscopy, DNA isolation and analysis, and western blotting (immunoblotting) to follow apoptosis in cultured human cells. Students working in teams chemically induce apoptosis, and harvest, process, and analyze cells, using their data to determine the order of events during apoptosis. We, as instructors, expose the students to an environment closely simulating what they would encounter in an active cell or molecular biology research laboratory by having students coordinate and perform multiple tasks simultaneously and by having them experience experimental design using current literature, data interpretation, and analysis to answer a single question. Students are assessed by examination of laboratory notebooks for completeness of experimental protocols and analysis of results and for completion of an assignment that includes questions pertaining to data interpretation and apoptosis.

Image analysis tools are currently in use for a variety of applications in the fields of forensics and security printing. Objective, quantitative data allow for characterization and comparison of a variety of image attributes.In forensics, document examiners can use quantitative image analysis systems in authentication, detection and analysis of forgeries, counterfeits and altered documents. Statistical analysis enables population characterization that can help in forgery detection and supports the potential for source identification. In security printing, image analysis tools can be applied to measure the quality of printed output and assess wear for bank notes.Flexible hardware options allow for a broad application of image quality measurements in a variety of image analysis situations. Camera and scanner-based image quality systems can be used for quantitative analysis of image content. A laser-based system can be used for height and depth profiling. Oblique angle illumination and scanning can be used for impression detection. A flatbed scanner can be used in transmission mode for the evaluation of bank note wear and for watermark analysis.In this paper, we will be discussing the use of a variety of hardware configurations for image analysis in forensics and security printing.

Tissue-based investigations can prove challenging due to complex tissue architecture and heterogeneous biomarker expression, visual and cognitive “traps” that affect interpretive precision, and subjective assessments that affect reproducibility. A major concern is that these challenges could increase the risk of failure for therapeutic/diagnostic co-development and clinical use, as the biomarker measurements continue to increase in complexity and require increasingly precise diagnostic cut-points. Image analysis tools have been developed to overcome some of the challenges for conventional anatomic pathology practices, capitalizing on the objectivity and computational power of a digital platform. A computer, however, lacks the cognitive ability and experience of a human to interpret tissue architecture and context. Flagship Biosciences’ computational Tissue Analysis (cTA™) platform integrates the power of our tissue Image Analysis (tIA™) technology with the contextual experience of an anatomic pathologist to produce robust, precise, quantitative results that demonstrate biomarker content in the tissue context. Flagship Biosciences envisions the integration of our cTA™ technology into a computer-aided clinical pathology workflow as a method to improve the precision of scoring for even some of the most challenging tissue-based biomarker measurements. In a proof-of-concept study, we evaluated the performance of manual versus digital scoring approaches in a cohort of non-small cell lung carcinoma (NSCLC) samples stained with the IHC protocol for the PD-L1 PharmDx 28-8 complementary diagnostic. A comparison of the 2 modalities demonstrated that in nearly all cases, the within sample standard deviation of the cTA™ digital score results was less than the manual score (median inter-pathologist %CVs were reduced from 124.9% to 7.8% and intra-pathologists from 65.4% to 7.6% for manual and digital scores, respectively). As an additional exploratory examination, the effect of heterogeneity on PD-L1 interpretation was also investigated. Pathologists evaluated the same whole tissue slides within 5 high powered fields (HPFs) using both manual and cTA™ -derived scoring. Results demonstrated that the use of cTA™ provides improves agreement between HPF and whole slide assessments (absolute difference between the manual scores from HPF to whole slide were larger than the absolute differences for the digitally derived scores, at 3.14% and 8.27%, respectively). Taken together, these studies demonstrate that the use of cTA™ can significantly reduce variability in PD-L1 scoring, as compared to a manual scoring approach. Citation Format: Staci Kearney, Joshua Black, Famke Aeffner, Joshua Black, Luke Pratte, Joseph Krueger. Evaluating benefits of PD-L1 image analysis for the clinical setting [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4582. doi:10.1158/1538-7445.AM2017-4582

Nonequilibrium gasdynamics processes of importance to hypersonic aerobraking vehicles are reviewed. Recent improvements in understanding these phenomena and in the detailed numerical modeling of these processes will be discussed. The paper concludes by describing the extent to which these models have been incorporated into multidimensional computational fluid dynamics (CFD) computer codes.

P-glycoprotein is implicated in clinical drug resistance; thus, rapid quantitative analysis of its expression and activity is of paramout importance to the design and success of novel therapeutics. The scope for the application of quantitative imaging and image analysis tools in this field is reported here at "proof of concept" level. P-glycoprotein expression was utilized as a model for quantitative immunofluorescence and subsequent spatial intensity distribution analysis (SpIDA). Following expression studies, p-glycoprotein inhibition as a function of verapamil concentration was assessed in two cell lines using live cell imaging of intracellular Calcein retention and a routine monolayer fluorescence assay. Intercellular and sub-cellular distributions in the expression of the p-glycoprotein transporter between parent and MDR1-transfected Madin-Derby Canine Kidney cell lines were examined. We have demonstrated that quantitative imaging can provide dose-response parameters while permitting direct microscopic analysis of intracellular fluorophore distributions in live and fixed samples. Analysis with SpIDA offers the ability to detect heterogeniety in the distribution of labeled species, and in conjunction with live cell imaging and immunofluorescence staining may be applied to the determination of pharmacological parameters or analysis of biopsies providing a rapid prognostic tool.

The finite Zak transform: An efficient tool for image representation and analysis