Search

Summary: FunImageJ is a Lisp framework for scientific image processing built upon the ImageJ software ecosystem. The framework provides a natural functional-style for programming, while accounting for the performance requirements necessary in big data processing commonly encountered in biological image analysis., Availability and Implementation: Freely available plugin to Fiji (http://fiji.sc/#download). Installation and use instructions available at http://imagej.net/FunImageJ., Contact: kharrington@uidaho.edu., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author(s) 2017. Published by Oxford University Press.)

Summary: State-of-the-art light and electron microscopes are capable of acquiring large image datasets, but quantitatively evaluating the data often involves manually annotating structures of interest. This process is time-consuming and often a major bottleneck in the evaluation pipeline. To overcome this problem, we have introduced the Trainable Weka Segmentation (TWS), a machine learning tool that leverages a limited number of manual annotations in order to train a classifier and segment the remaining data automatically. In addition, TWS can provide unsupervised segmentation learning schemes (clustering) and can be customized to employ user-designed image features or classifiers., Availability and Implementation: TWS is distributed as open-source software as part of the Fiji image processing distribution of ImageJ at http://imagej.net/Trainable_Weka_Segmentation ., Contact: ignacio.arganda@ehu.eus., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com)

Summary: ImageJ-MATLAB is a lightweight Java library facilitating bi-directional interoperability between MATLAB and ImageJ. By defining a standard for translation between matrix and image data structures, researchers are empowered to select the best tool for their image-analysis tasks., Availability and Implementation: Freely available extension to ImageJ2 ( http://imagej.net/Downloads ). Installation and use instructions available at http://imagej.net/MATLAB_Scripting. Tested with ImageJ 2.0.0-rc-54 , Java 1.8.0_66 and MATLAB R2015b., Contact: eliceiri@wisc.edu., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com)

Anthocyanins are flavonoid pigments synthesized in the cytoplasm and stored inside vacuoles. Many plant species accumulate densely packed, 3- to 10-μm diameter anthocyanin deposits called anthocyanin vacuolar inclusions (AVIs). Despite their conspicuousness and importance in organ coloration, the origin and nature of AVIs have remained controversial for decades. We analyzed AVI formation in cotyledons of different Arabidopsis thaliana genotypes grown under anthocyanin inductive conditions and in purple petals of lisianthus (Eustoma grandiorum). We found that cytoplasmic anthocyanin aggregates in close contact with the vacuolar surface are directly engulfed by the vacuolar membrane in a process reminiscent of microautophagy. The engulfed anthocyanin aggregates are surrounded by a single membrane derived from the tonoplast and eventually become free in the vacuolar lumen like an autophagic body. Neither endosomal/prevacuolar trafficking nor the autophagy ATG5 protein is involved in the formation of AVIs. In Arabidopsis, formation of AVIs is promoted by both an increase in cyanidin 3-O-glucoside derivatives and by depletion of the glutathione S-transferase TT19. We hypothesize that this novel microautophagy mechanism also mediates the transport of other flavonoid aggregates into the vacuole., (© 2015 American Society of Plant Biologists. All rights reserved.)

In prostate cancer, bone is a frequent site of metastasis; however, the molecular mechanisms of this tumor tropism remain unclear. Here, we integrate a microfluidic coculture platform with multi-photon imaging based techniques to assess both phenotypic cell behavior and FAD fluorescence intensity and fluorescence lifetime in the same cell. This platform combines two independent assays normally performed with two different cell populations into a single device, allowing us to simultaneously assess both phenotypic cell behavior and enzyme activity. We observed that the osteotropic prostate cancer cell line (C4-2B), when in a coculture with bone marrow stromal cells (MC3T3-E1), has increased protrusive phenotype and increased total and protein-bound FAD compared to its parent cell line (LNCaP). We hypothesized that an increase in ROS-generating APAO activity may be responsible for these effects, and found that the effects were decreased in the presence of the antioxidant N-Acetyl Cysteine (NAC). This suggests that an ROS-related signaling mechanism at the bone metastatic site may be correlated with and play a role in increased invasion of metastasizing prostate cancer cells. The studies performed using this combined platform will lead to new insights into the mechanisms that drive prostate cancer metastasis.

Embryoid bodies (EBs) are large (>100 μm) 3D microtissues composed of stem cells, differentiating cells and extracellular matrix (ECM) proteins that roughly recapitulate early embryonic development. EBs are widely used as in vitro model systems to study stem cell differentiation and the complex physical and chemical interactions contributing to tissue development. Though much has been learned about differentiation from EBs, the practical and technical difficulties of effectively probing and properly analyzing these 3D microtissues has limited their utility and further application. We describe advancement of a technology platform developed in our laboratory, multiphoton flow cytometry (MPFC), to detect and sort large numbers of intact EBs based on size and fluorescent reporters. Real-time and simultaneous measurement of size and fluorescence intensity are now possible, through the implementation of image processing algorithms in the MPFC software. We applied this platform to purify populations of EBs generated from murine induced pluripotent stem (miPS) cells exhibiting enhanced potential for cardiomyocyte differentiation either as a consequence of size or expression of NKX2-5, a homeodomain protein indicative of precardiac cells. Large EBs (330-400 μm, diameter) purified soon after EB formation showed significantly higher potential to form cardiomyocytes at later time points than medium or small EBs. In addition, EBs expressing NKX2-5 soon after EB formation were more likely to form beating areas, indicative of cardiomyocyte differentiation, at later time points. Collectively, these studies highlight the ability of the MPFC to purify EBs and similar microtissues based on preferred features exhibited at the time of sorting or on features indicative of future characteristics or functional capacity.

Ligament healing of a grade III injury (i.e., a complete tear) involves a multifaceted chain of events that forms a neoligament, which is more scar-like in character than the native tissue. The remodeling process may last months or even years with the injured ligament never fully recovering pre-injury mechanical properties. With tissue engineering and regenerative medicine, understanding the normal healing process in ligament and quantifying it provide a basis to create and assess innovative treatments. Ligament fibroblasts produce a number of extracellular matrix (ECM) components, including collagen types I and III, decorin and fibromodulin. Using a combination of advanced histology, molecular biology, and nonlinear optical imaging approaches, the early ECM events during ligament healing have been better characterized and defined. First, the dynamic changes in ECM factors after injury are shown. Second, the factors associated with creeping substitution are identified. Finally, a method to quantify collagen organization is developed and used. Each ECM factor described herein as well as the temporal quantification of fiber organization helps elucidate the complexity of ligament healing.

Detection and tracking of stem cell state are difficult due to insufficient means for rapidly screening cell state in a noninvasive manner. This challenge is compounded when stem cells are cultured in aggregates or three-dimensional (3D) constructs because living cells in this form are difficult to analyze without disrupting cellular contacts. Multiphoton laser scanning microscopy is uniquely suited to analyze 3D structures due to the broad tunability of excitation sources, deep sectioning capacity, and minimal phototoxicity but is throughput limited. A novel multiphoton fluorescence excitation flow cytometry (MPFC) instrument could be used to accurately probe cells in the interior of multicell aggregates or tissue constructs in an enhanced-throughput manner and measure corresponding fluorescent properties. By exciting endogenous fluorophores as intrinsic biomarkers or exciting extrinsic reporter molecules, the properties of cells in aggregates can be understood while the viable cellular aggregates are maintained. Here we introduce a first generation MPFC system and show appropriate speed and accuracy of image capture and measured fluorescence intensity, including intrinsic fluorescence intensity. Thus, this novel instrument enables rapid characterization of stem cells and corresponding aggregates in a noninvasive manner and could dramatically transform how stem cells are studied in the laboratory and utilized in the clinic.

Unlabelled: There is a strong and growing need in the biology research community for accurate, automated image analysis. Here, we describe CellProfiler 2.0, which has been engineered to meet the needs of its growing user base. It is more robust and user friendly, with new algorithms and features to facilitate high-throughput work. ImageJ plugins can now be run within a CellProfiler pipeline., Availability and Implementation: CellProfiler 2.0 is free and open source, available at http://www.cellprofiler.org under the GPL v. 2 license. It is available as a packaged application for Macintosh OS X and Microsoft Windows and can be compiled for Linux., Contact: anne@broadinstitute.org, Supplementary Information: Supplementary data are available at Bioinformatics online.

The transition of ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC) is a critical step in breast cancer progression. We introduce a simple microfluidic 3D compartmentalized system in which mammary epithelial cells (MCF-DCIS) are co-cultured with human mammary fibroblasts (HMFs), which promotes a transition from DCIS to IDC in vitro. The model enables control of both spatial (distance-dependence) and temporal (transition from larger clusters) aspects within the microenvironment, allowing recapitulation of the in vivo environment in ways not practical with existing experimental models. When HMFs were cultured some distance (0.5-1.5 mm) from the MCF-DCIS cells, we observed an initial morphological change, suggesting soluble factors can begin the transition. However, cell-cell contact with HMFs allowed the MCF-DCIS cells to complete the transition to invasion. Uniquely, the compartmentalized platform enables the analysis of the intrinsic second harmonic generation signal of collagen, providing a label-free quantitative analysis of DCIS-associated collagen remodeling. The arrayed microchannel-based model is compatible with existing infrastructure and, for the first time, provides a cost effective approach to test for inhibitors of pathways involved in DCIS progression to IDC allowing a screening approach to the identification of potential therapeutic targets. Importantly, the model can be easily adapted and generalized to a variety of cell-cell signaling studies.

We present an in vitro model of human skin that, together with nonlinear optical microscopy, provides a useful system for characterizing morphological and structural changes in a living skin tissue microenvironment due to changes in oxygen status and proteolytic balance. We describe for the first time the effects of chronic oxygen deprivation on a bioengineered model of human interfollicular epidermis. Histological analysis and multiphoton imaging revealed a progressively degenerating ballooning phenotype of the keratinocytes that manifested after 48 h of hypoxic exposure. Multiphoton images of the dermal compartment revealed a decrease in collagen structural order. Immunofluorescence analysis showed changes in matrix metalloproteinase (MMP)-2 protein spatial localization in the epidermis with a shift to the basal layer, and loss of Ki67 expression in proliferative basal cells after 192 h of hypoxic exposure. Upon reoxygenation MMP-2 mRNA levels showed a biphasic response, with restoration of MMP-2 levels and localization. These results indicate that chronic oxygen deprivation causes an overall degeneration in tissue architecture, combined with an imbalance in proteolytic expression and a decrease in proliferative capacity. We propose that these tissue changes are representative of the ischemic condition and that our experimental model system is appropriate for addressing mechanisms of susceptibility to chronic wounds.

Nonlinear optical imaging techniques such as multiphoton and second harmonic generation (SHG) microscopy used in conjunction with novel signal analysis techniques such as spectroscopic and fluorescence excited state lifetime detection have begun to be used widely for biological studies. This is largely due to their promise to noninvasively monitor the intracellular processes of a cell together with the cell's interaction with its microenvironment. Compared to other optical methods these modalities provide superior depth penetration and viability and have the additional advantage in that they are compatible technologies that can be applied simultaneously. Therefore, application of these nonlinear optical approaches to the study of breast cancer holds particular promise as these techniques can be used to image exogeneous fluorophores such as green fluorescent protein as well as intrinsic signals such as SHG from collagen and endogenous fluorescence from nicotinamide adenine dinucleotide or flavin adenine dinucleotide. In this article the application of multiphoton excitation, SHG, and fluorescence lifetime imaging microscopy to relevant issues regarding the tumor-stromal interaction, cellular metabolism, and cell signaling in breast cancer is described. Furthermore, the ability to record and monitor the intrinsic fluorescence and SHG signals provides a unique tool for researchers to understand key events in cancer progression in its natural context.

The use of multifocal-plane, time-lapse recordings of living specimens has allowed investigators to visualize dynamic events both within ensembles of cells and individual cells. Recordings of such four-dimensional (4D) data from digital optical sectioning microscopy produce very large data sets. We describe a wavelet-based data compression algorithm that capitalizes on the inherent redunancies within multidimensional data to achieve higher compression levels than can be obtained from single images. The algorithm will permit remote users to roam through large 4D data sets using communication channels of modest bandwidth at high speed. This will allow animation to be used as a powerful aid to visualizing dynamic changes in three-dimensional structures.