Mr. Green Genes: MetaGFP(TM) from Universal Imaging Corporation Allows Quantification of Fluorescent Probes

The use of green fluorescent protein (GFP) from the jellyfish Aequorea Victoria to monitor gene expression both in situ and in vivo has grabbed the biotechnology spotlight since this application was reported almost four years ago (M. Chalfie et al., Science 263:802-805, 1994). Biologists could ask for no simpler tool; GFP's bright green fluorescence stems from its inherent chromophore structure and needs no substrate or cofactors, only exposure to UV or blue light. Along with wild-type GFP, several optimized mutants of this protein are on the market. This being the case, researchers in a variety of biological arenas are using this protein to study genes in numerous organisms, from bacteria and yeast to plants and animals. In November 1997, Universal Imaging Corporation released MetaGFP Version 3.0, a unique software package for the collection and analysis of images from GFP and multiprobe fluorescence experiments. Co-localization of two fluorescent probes Prior to developing the software, the designers of MetaGFP interviewed numerous researchers in a variety of disciplines using GFP and discovered many did not have experience with fluorescent microscopy and low-light-level imaging. With this in mind, MetaGFP was designed to be easier to use than any other imaging system on the market yet valuable to both beginners and experienced users of the technology. Impressed with the features of this new software package is James Sabry, formerly with Stanford University and now President and Chief Technology Officer of Cytokinetics, Inc. (San Francisco, Calif.). "The real challenge with fluorescent probes is how to quantify an image as actual data. MetaGFP lets investigators acquire, store, and more importantly, quantify these images," remarked Sabry, who has used MetaGFP with a digital cooled CCD. Sabry is researching mutations in the molecular motor myosin II and characterizing how these mutations affect the function and cellular localization of the protein. MetaGFP is flexible, supporting the most commonly used imaging equipment and microscope accessories. It is optimized for taking time-lapse images of living cells using two fluorescent wavelengths and one transmitted light mode. This allows researchers to potentially identify a specified protein, monitor a subcellular compartment of another protein, and monitor the boundaries of the cell or subcellular structures. As an example, a protein would appear in green, nuclear stain fluorescence would appear in blue, and overlap between the two would appear as aqua, while nonfluorescent cell constituents would be various shades of gray. MetaGFP takes control of the microscope and camera for image collection, collects the images, and analyzes the quantity of protein present, where it is present, and how much localizes with other fluorescents probes of interest. MetaGFP automates image collection by controlling the illumination (filter wheels, shutters, monochromateors) and camera (video, integrating, cooled digital CCD). Rhodamine-labeled microtubules and a flurescent ER marker in newt lung cells. Image courtesy of Clare Waterman-Storer and Ed Salmon at UNC-Chapel Hill. Several quantitative and qualitative image analysis techniques are made possible with MetaGFP. For example, fluorescent images are automatically overlaid with the transmitted light images into 24-bit color or monochrome images, and color intensitites can be balanced to accentuate the areas in which fluorescent probes overlap. MetaGFP lets users define numerous regions and on the image can measure the area, integrated intensity, or average intensity within each region. Overlap of fluorescence can also be quantified. Graphs of any of these values can be generated during image acquisition or later when the experiment is reviewed. MetaGFP is also capable of calculating a correlation coefficient and generating a correlation plot of two fluorescent probes. Images are displayed using a new movie feature or by exporting them into any standard presentation graphics software. MetaGFP uses the 32-bit capabilities of Microsoft Windows 95TM including long, descriptive file names, faster operation, and advanced printing options. For more information contact Universal Imaging Corporation at 1-610-344-9410 or www.image1.com

Mr. Green Genes: MetaGFP(TM) from Universal Imaging Corporation Allows Quantification of Fluorescent Probes

The Scientist Staff

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