[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Fiji is a distribution of the popular open-source software ImageJ focused on biological-image analysis. Fiji uses modern software engineering practices to combine powerful software libraries with a broad range of scripting languages to enable rapid prototyping of image-processing algorithms. Fiji facilitates the transformation of new algorithms into ImageJ plugins that can be shared with end users through an integrated update system. We propose Fiji as a platform for productive collaboration between computer science and biology research communities.
[Show abstract][Hide abstract] ABSTRACT: 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.
Microscopy and Microanalysis 08/2011; 17(4):540-54. DOI:10.1017/S1431927610000280 · 1.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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: email@example.com SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
[Show abstract][Hide abstract] ABSTRACT: Data sharing is important in the biological sciences to prevent duplication of effort, to promote scientific integrity, and to facilitate and disseminate scientific discovery. Sharing requires centralized repositories, and submission to and utility of these resources require common data formats. This is particularly challenging for multidimensional microscopy image data, which are acquired from a variety of platforms with a myriad of proprietary file formats (PFFs). In this paper, we describe an open standard format that we have developed for microscopy image data. We call on the community to use open image data standards and to insist that all imaging platforms support these file formats. This will build the foundation for an open image data repository.
The Journal of Cell Biology 05/2010; 189(5):777-82. DOI:10.1083/jcb.201004104 · 9.83 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Recently, new non-invasive imaging methods have been developed and applied to cellular and animal mammary models that have enabled breast cancer researchers to track key players and events in mammary metastasis. Noninvasive nonlinear optical methods such as multiphoton laser scanning microscopy (MPLSM), Fluorescence Lifetime Microscopy (FLIM) and second harmonic generation (SHG) imaging provide an unrivaled ability for obtaining high-resolution images from deep within tissue that can be exploited in the quest to understand breast cancer progression. These optical methods can add greatly to our knowledge of cancer progression by allowing key processes to be non-invasively imaged such as metabolism (on the basis of free and bound NADH detection via FLIM) and interactions with the extracellular matrix (SHG imaging of collagen). In this short application note we present a survey of our latest optical and computational efforts to study intrinsic fluorescence in breast cancer models. In particular we present the latest development in our SLIM Plotter application, an open source visualization program for interactive visualization and inspection of combined spectral lifetime (SLIM) data.
Conference proceedings: ... Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Conference 01/2009; 2009:4077-80. DOI:10.1109/IEMBS.2009.5334523
[Show abstract][Hide abstract] ABSTRACT: Multiphoton laser scanning microscopy (MPLSM) utilizing techniques such as multiphoton excitation (MPE), second harmonic generation (SHG), and multiphoton fluorescence lifetime imaging and spectral lifetime imaging (FLIM and SLIM, respectively) are greatly expanding the degree of information obtainable with optical imaging in biomedical research. The application of these nonlinear optical approaches to the study of breast cancer holds particular promise. These noninvasive, multidimensional techniques are well suited to image exogenous fluorophores that allow relevant questions regarding protein localization and signaling to be addressed both in vivo and in vitro. Furthermore, MPLSM imaging of endogenous signals from collagen and fluorophores such as nicotinamide adenine dinucleotide (NADH) or flavin adenine dinucleotide (FAD), address important questions regarding the tumor-stromal interaction and the physiologic state of the cell. We demonstrate the utility of multimodal MPE/SHG/FLIM for imaging both exogenous and/or endogenous fluorophores in mammary tumors or relevant 3-D systems. Using SLIM, we present a method for imaging and differentiating signals from multiple fluorophores that can have overlapping spectra via SLIM Plotter-a computational tool for visualizing and analyzing large spectral-lifetime data sets.
[Show abstract][Hide abstract] ABSTRACT: Mammographically dense breast tissue is one of the greatest risk factors for developing breast carcinoma. Despite the strong clinical correlation, breast density has not been causally linked to tumorigenesis, largely because no animal model has existed for studying breast tissue density. Importantly, regions of high breast density are associated with increased stromal collagen. Thus, the influence of the extracellular matrix on breast carcinoma development and the underlying molecular mechanisms are not understood.
To study the effects of collagen density on mammary tumor formation and progression, we utilized a bi-transgenic tumor model with increased stromal collagen in mouse mammary tissue. Imaging of the tumors and tumor-stromal interface in live tumor tissue was performed with multiphoton laser-scanning microscopy to generate multiphoton excitation and spectrally resolved fluorescent lifetimes of endogenous fluorophores. Second harmonic generation was utilized to image stromal collagen.
Herein we demonstrate that increased stromal collagen in mouse mammary tissue significantly increases tumor formation approximately three-fold (p < 0.00001) and results in a significantly more invasive phenotype with approximately three times more lung metastasis (p < 0.05). Furthermore, the increased invasive phenotype of tumor cells that arose within collagen-dense mammary tissues remains after tumor explants are cultured within reconstituted three-dimensional collagen gels. To better understand this behavior we imaged live tumors using nonlinear optical imaging approaches to demonstrate that local invasion is facilitated by stromal collagen re-organization and that this behavior is significantly increased in collagen-dense tissues. In addition, using multiphoton fluorescence and spectral lifetime imaging we identify a metabolic signature for flavin adenine dinucleotide, with increased fluorescent intensity and lifetime, in invading metastatic cells.
This study provides the first data causally linking increased stromal collagen to mammary tumor formation and metastasis, and demonstrates that fundamental differences arise and persist in epithelial tumor cells that progressed within collagen-dense microenvironments. Furthermore, the imaging techniques and signature identified in this work may provide useful diagnostic tools to rapidly assess fresh tissue biopsies.
BMC Medicine 04/2008; 6(1):11. DOI:10.1186/1741-7015-6-11 · 7.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Effective data analysis of the modern biological microscopy data set often necessitates a variety of different analysis strategies, and this often means the biologist may need to use a combination of software tools both commercial and often-times open source. To facilitate this process, there needs to be knowledge of what the approaches are and also practical ways of sharing this data in a nonproprietary way. Thus, for users of open source and commercial software, it is important to have common approaches for multidimensional data analysis that can be run in different software packages and still be effectively compared. Projects like the Open Microscopy Environment, which aim to allow data sharing between open source client tools like ImageJ and VisBio, and commercial packages like Volocity and Imaris via the XML data model are a needed first step in providing a framework or infrastructure for microscopy analysis. As the field has gotten more quantitative in its approaches, this need has only increased with the necessity of having a way to represent key attributes of the data in an open manner.
[Show abstract][Hide abstract] ABSTRACT: Live cell imaging has been greatly advanced by the recent development of new fluorescence microscopy-based methods such as multiphoton laser-scanning microscopy, which can noninvasively image deep into live specimens and generate images of extrinsic and intrinsic signals. Of recent interest has been the development of techniques that can harness properties of fluorescence, other than intensity, such as the emission spectrum and excited state lifetime of a fluorophore. Spectra can be used to discriminate between fluorophores, and lifetime can be used to report on the microenvironment of fluorophores. We describe a novel technique-combined spectral and lifetime imaging-which combines the benefits of multiphoton microscopy, spectral discrimination, and lifetime analysis and allows for the simultaneous collection of all three dimensions of data along with spatial and temporal information.
[Show abstract][Hide abstract] ABSTRACT: Over the last 50 years modern cell biology has been driven by the development of powerful imaging techniques. In particular, new developments in light microscopy that provide the potential to image the dynamics of biological events have had significant impact. Optical sectioning techniques allow three-dimensional information to be obtained from living specimens noninvasively. When used with multimodal fluorescence microscopy, advanced optical sectioning techniques provide multidimensional image data that can reveal information not only about the changing cytoarchitecture of a cell but also about its physiology. These additional dimensions of information, although providing powerful tools, also pose significant visualization challenges to the investigator. Particularly in the current postgenomic era there is a greater need than ever for the development of effective tools for image visualization and management. In this review we discuss the visualization challenges presented by multidimensional imaging and describe three open-source software programs being developed to help address these challenges: ImageJ, the Open Microscopy Environment, and VisBio.
Photochemistry and Photobiology 09/2005; 81(5):1116-22. DOI:10.1562/2004-11-22-IR-377 · 2.27 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Combining a flexible data model and distributed objects, they support the sharing of data, visualizations, and user interfaces among multiple data sources, computers, and scientific disciplines.
Communications of the ACM 03/2005; 48(3):98-104. DOI:10.1145/1047671.1047676 · 3.62 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: New laser scanning microscopy techniques enable biologists to acquire larger, more complex image datasets. Emerging imaging modalities such as multispectral, harmonic, and fluorescence lifetime can generate data with six or more dimensions; however, existing software is not well suited to the visualization or analysis of such data. To address these concerns, we have developed VisBio, an application and toolkit for visualization and analysis of multidimensional, biological image data of any dimensionality.
[Show abstract][Hide abstract] ABSTRACT: The scientific world is evolving to require more collaboration among different institutions and disciplines. Understanding long-term changes in the Earth environment, for example, requires models that integrate disciplines such as meteorology, oceanography, hydrology (rivers and groundwater), soil science and geology. During the past 15 years, scientists have started sharing data using FTP and software on the Internet, but collaborative work and more routine data sharing require a new kind of scientific software.
Communications of the ACM 04/2002; 45(4ve):160-170. DOI:10.1145/505248.506005 · 3.62 Impact Factor