An Integrated Approach to Fluorescence Imaging

Image: Courtesy of TILL Photonics TILLvisDECO in action. Image before (middle) and after (right) deconvolution. The quality of the data obtained from fluorescence imaging studies depends on the system of components used to acquire, analyze, and process the fluorescent images. As an alternative to building a system piecemeal, using components from several vendors, TILL Photonics of Gräfelfing, Germany, offers its fully integrated TILLvisION system and software modules for fluorescence imaging. As the heart of the TILL Photonics' fluorescence imaging product line, the TILLvisION system includes five integrated components: the Polychrome IV excitation light source, an interline frame-transfer camera, a DSP-based imaging system controller board that maintains exact timing during the experiment, TILLvisION 4.0 software, and an imaging workstation. The basic TILLvisION system offers two main advantages: customizability and speed. Scientists can use the system with Leica, Nikon, Olympus, and Zeiss fluorescent microscopes. The company designs custom epifluorescence condensers to match each microscope's optics. The Polychrome IV illumination system can switch its output wavelength in three milliseconds, and the TILLvisION 4.0 software can acquire images at up to 30 frames per second. According to John Zemek, Director of TILL Photonics, Inc., TILL's Eugene, Ore.-based US subsidiary, such speed is critical for fluorescence imaging of living systems. For example, Zemek says the Polychrome IV's rapid wavelength switching is ideally suited for analyzing calcium ion concentration using the fura-2 indicator excited alternatively at two wavelengths. By coupling the customized condenser to the Polychrome IV high-speed scanning monochromator, the TILLvisION system optimizes the light output to the specimen plane for both brightness and homogeneity. The TILLvisION 4.0 software brings these two features together with a full range of features for experiment setup, image acquisition, analysis, data export, and image processing. In addition to the core TILLvisION 4.0 application that comes bundled with the complete TILLvisION system, TILL Photonics also offers three add-on software modules. The first, TILLvisTRAC, tracks particles as they move through living cells. After images are acquired at a high rate with TILLvisION 4.0, an anisotropic diffusion filter is applied and an edge-based segmentation algorithm detects and isolates the particles. The software then runs a fuzzy-logic algorithm on the filtered information to produce quantitative evaluations of particle trajectory and velocity, as well as three-dimensional profiles that show movement in two directions versus time. Researchers can use this module to, for example, analyze trafficking of secretory membrane vesicles, or to watch as differently labeled vesicles move towards each other and coalesce. A second module, TILLvisFRET, measures the effects of fluorescent resonance energy transfer (FRET). In FRET, energy transfer occurs between a donor and an acceptor fluorophore-labeled molecule when they come into close proximity, affecting the fluorescent signal that each labeled molecule emits. TILLvisFRET analyzes a single sample simultaneously at multiple wavelengths through the use of a beam splitter module placed in the emission path between the microscope and camera. In this way, the system simultaneously collects images at two or four different wavelengths, which can then be examined separately or overlaid. One application of this module: Scientists can measure the distance between two interacting molecules, such as a receptor and its ligand. FRET analysis surpasses the spatial resolution capabilities of standard fluorescent imaging techniques, which typically measure distances on the order of 0.2 µm. The third module, TILLvisDECO, removes the blurring introduced by out-of-focus light from fluorescence images. Using a piezoelectric objective motor, this module measures the characteristic impulse response of the optical system, known as the point-spread function. From these data the software computes a filter that it then applies to the acquired images to improve their quality. The calculation of the 'deconvolved' corrected image requires only minutes, as opposed to standard filter calculations that require several hours. The TILLvisION system, which includes a Dell Pentium 4 computer, sells for $48,000 (US). This price includes installation, training, and startup in the laboratory. The additional software modules are priced at $7,000 for TILLvisTRAC; $9,000 for TILLvisFRET with its beam-splitter module; and $10,000 for TILLvisDECO and the piezo Z-drive device for fine adjustment of focus. --Linda Raab

An Integrated Approach to Fluorescence Imaging

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Image: Courtesy of TILL Photonics
TILLvisDECO in action. Image before (middle) and after (right) deconvolution.