Metadata matters: access to image data in the real world

Linkert, Melissa; Rueden, Curtis; Allan, Chris; Burel, Jean‐Marie; Moore, W. B.; Patterson, Andrew; Loranger, Brian; Moore, Josh; Neves, Carlos; MacDonald, Donald; Tarkowska, Aleksandra; Sticco, J Caitlin; Hill, Emma; Rossner, Mike; Eliceiri, Kevin W.; Swedlow, Jason R.

doi:10.1083/jcb.201004104

Cited by 928 publications

(810 citation statements)

References 18 publications

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“…The mica surface was prepared according to the protocol presented here. To estimate optical aberrations, full width at half maximum (FWHM) was measured and averaged over the 20 point spread function (PSF) using Mosaic 2D PSF Size ImageJ plugin 16,17 . The measured FWHM for glass and mica were 441nm and 464nm respectively (Figure 1).…”

Section: Representative Resultsmentioning

confidence: 99%

Preparation of Mica Supported Lipid Bilayers for High Resolution Optical Microscopy Imaging

Matysik

Kraut

2014

JoVE

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Supported lipid bilayers (SLBs) are widely used as a model for studying membrane properties (phase separation, clustering, dynamics) and its interaction with other compounds, such as drugs or peptides. However SLB characteristics differ depending on the support used.Commonly used techniques for SLB imaging and measurements are single molecule fluorescence microscopy, FCS and atomic force microscopy (AFM). Because most optical imaging studies are carried out on a glass support, while AFM requires an extremely flat surface (generally mica), results from these techniques cannot be compared directly, since the charge and smoothness properties of these materials strongly influence diffusion. Unfortunately, the high level of manual dexterity required for the cutting and gluing thin slices of mica to the glass slide presents a hurdle to routine use of mica for SLB preparation. Although this would be the method of choice, such prepared mica surfaces often end up being uneven (wavy) and difficult to image, especially with small working distance, high numerical aperture lenses. Here we present a simple and reproducible method for preparing thin, flat mica surfaces for lipid vesicle deposition and SLB preparation. Additionally, our custom made chamber requires only very small volumes of vesicles for SLB formation. The overall procedure results in the efficient, simple and inexpensive production of high quality lipid bilayer surfaces that are directly comparable to those used in AFM studies. Video LinkThe video component of this article can be found at

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Section: Representative Resultsmentioning

confidence: 99%

Preparation of Mica Supported Lipid Bilayers for High Resolution Optical Microscopy Imaging

Matysik

Kraut

2014

JoVE

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“…Following acquisition, a FIJI plugin 22 was used to stitch the images together based on OME-XML metadata. 23,24 Two-photon excited fluorescence images were also acquired without filtering in selected regions of interest.…”

Section: Multiphoton Microscopymentioning

confidence: 99%

Periductal stromal collagen topology of pancreatic ductal adenocarcinoma differs from that of normal and chronic pancreatitis

et al. 2015

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Pancreatic ductal adenocarcinoma continues to be one of the most difficult diseases to manage with one of the highest cancer mortality rates. This is due to several factors including nonspecific symptomatology and subsequent diagnosis at an advanced stage, aggressive metastatic behavior that is incompletely understood, and limited response to current therapeutic regimens. As in other cancers, there is great interest in studying the role of the tumor microenvironment in pancreatic ductal adenocarcinoma and whether components of this environment could serve as research and therapeutic targets. In particular, attention has turned toward the desmoplastic collagen-rich pancreatic ductal adenocarcinoma stroma for both biological and clinical insight. In this study, we used quantitative second harmonic generation microscopy to investigate stromal collagen organization and structure in human pancreatic ductal adenocarcinoma pathology tissues compared with non-neoplastic tissues. Collagen topology was characterized in whole-tissue microarray cores and at specific pathology-annotated epithelial-stroma interfaces representing 241 and 117 patients, respectively. We quantitatively demonstrate that a unique collagen topology exists in the periductal pancreatic ductal adenocarcinoma stroma. Specifically, collagen around malignant ducts shows increased alignment, length, and width compared with normal ducts and benign ducts in a chronic pancreatitis background. These findings indicate that second harmonic generation imaging can provide quantitative information about fibrosis that complements traditional histopathologic insights and can serve as a rich field for investigation into pathogenic and clinical implications of reorganized collagen as a pancreatic ductal adenocarcinoma disease marker. Pancreatic ductal adenocarcinoma is one of the most devastating human malignancies. It is currently the fourth leading cause of cancer death and projects to become the second leading cause by 2030. 1 The 5-year relative survival rate has remained in the single digits for decades. Pancreatic ductal adenocarcinoma is notoriously difficult to detect before an advanced, inoperable stage is reached due to nonspecific symptomatology and the retroperitoneal location of the pancreas, which makes palpation or routine biopsy of suspicious masses difficult. Recent advances in the sensitivity and specificity of preoperative imaging modalities such as computer tomography, magnetic resonance imaging, positron emission tomography, endoscopic ultrasonography, and endoscopic retrograde cholangiopancreatography have played an important role in enhancing pancreatic ductal adenocarcinoma diagnosis, detailing the relationship of lesions to nearby anatomical structures, and determining the course of management. 2 Despite these advances, only 10-15% of patients are diagnosed at a localized disease stage when surgical resection is possible as a potential curative therapy. However, postsurgical recurrence rates are high with 5-year survival rate of

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“…25 for analysis of FRAP movies using Nikon ND2 files. The current program reads OME-TIFF files 49 , because most file formats can readily be converted to OME-TIFF. A quantitative analysis of FRAP data is easiest and most accurate when bleaching is instantaneous, the bleached region is a circle, and bleaching during read-out is negligible.…”

Section: Suv-sbl Fusion Assay To Monitor Lipid and Content Release Simentioning

confidence: 99%

SNARE-mediated Fusion of Single Proteoliposomes with Tethered Supported Bilayers in a Microfluidic Flow Cell Monitored by Polarized TIRF Microscopy

Nikolaus

Karatekin

2016

JoVE

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In the ubiquitous process of membrane fusion the opening of a fusion pore establishes the first connection between two formerly separate compartments. During neurotransmitter or hormone release via exocytosis, the fusion pore can transiently open and close repeatedly, regulating cargo release kinetics. Pore dynamics also determine the mode of vesicle recycling; irreversible resealing results in transient, "kiss-and-run" fusion, whereas dilation leads to full fusion. To better understand what factors govern pore dynamics, we developed an assay to monitor membrane fusion using polarized total internal reflection fluorescence (TIRF) microscopy with single molecule sensitivity and ~15 msec time resolution in a biochemically well-defined in vitro system. Fusion of fluorescently labeled small unilamellar vesicles containing v-SNARE proteins (v-SUVs) with a planar bilayer bearing t-SNAREs, supported on a soft polymer cushion (t-SBL, t-supported bilayer), is monitored. The assay uses microfluidic flow channels that ensure minimal sample consumption while supplying a constant density of SUVs. Exploiting the rapid signal enhancement upon transfer of lipid labels from the SUV to the SBL during fusion, kinetics of lipid dye transfer is monitored. The sensitivity of TIRF microscopy allows tracking single fluorescent lipid labels, from which lipid diffusivity and SUV size can be deduced for every fusion event. Lipid dye release times can be much longer than expected for unimpeded passage through permanently open pores. Using a model that assumes retardation of lipid release is due to pore flickering, a pore "openness", the fraction of time the pore remains open during fusion, can be estimated. A soluble marker can be encapsulated in the SUVs for simultaneous monitoring of lipid and soluble cargo release. Such measurements indicate some pores may reseal after losing a fraction of the soluble cargo. Video LinkThe video component of this article can be found at

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Metadata matters: access to image data in the real world

Cited by 928 publications

References 18 publications

Preparation of Mica Supported Lipid Bilayers for High Resolution Optical Microscopy Imaging

Preparation of Mica Supported Lipid Bilayers for High Resolution Optical Microscopy Imaging

Periductal stromal collagen topology of pancreatic ductal adenocarcinoma differs from that of normal and chronic pancreatitis

SNARE-mediated Fusion of Single Proteoliposomes with Tethered Supported Bilayers in a Microfluidic Flow Cell Monitored by Polarized TIRF Microscopy

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