Fluorescence Illumination Lights Up Living Cells

A wide variety of cellular processes—including cell division, growth regulation, neural transmission, and ionic regulation—are regulated via transient shifts and/or steady-state shifts in the levels of molecules acting as signal transducers (for example, calcium flux, pH shift, and receptor binding). In order to investigate the mechanics of these complex multicomponent processes, bioscientists need to pinpoint and quantitate these event(s) in living cells and tissues. A variety of probes have been developed that allow the scientist to visualize and quantitate physiological events using fluorescence microscopy. Specific probes for calcium, for example, can distinguish changes in free versus sequestered calcium via shifts in the ratio of the probe’s fluorescence intensity at two different wavelengths. Ratio imaging microscopy allows the researcher to pinpoint the cellular location of the chemical shift utilizing low-light level imaging techniques, while ratio fluorescence microscopy allows one to quantitate the event with sensitive photo-detectors. However, numerous technical problems complicate these techniques, such as low signal-tonoise ratio detection, and time constraints on data acquisition. Photon Technology International Inc., based in Princeton, NJ., has developed a modular fluorescence illumination, detection, and data analysis system, designed to overcome these problems as well as offer the versatility for many different applications. The Deltascan illumination system employs a xenon arc source, which is collected with high-efficiency front surface optics. Light torn the source is alternatively chopped between two excitation monochromates (Czerny-Turner type) to generate continuously variable alternating wavelength light, whihc is subsequently recombined. The use of monochromators rather than filters allow, the scientist to determine the -excitation/emission spectra of the specimen in order to distinguish the fluorophore probe from contaminating fluorescent compounds. ‘The illumination system is interfaced with a fluorescence microscope (optionally available) via field lens resulting in Koehler illumination, a unique feature for this type of instrument. In addition, the illumination system is not mechanically coupled to the microscope, thus avoiding frame registration errors due to vibration from the light chopper, a major obstacle for ratio imaging. Fluorescent light resulting from specimen illumination is gathered either by dual channel photometers for quantitation (ratio measurement) or a low light level video camera for ratio imaging. The ratio measurement system offers rapid quantification of specimen fluorescence, while the imaging system provides for precise location of fluorescence in a particular region of the specimen. A second major feature in the design of the instrument, priced at $95,000, is the Imagescan image analysis system. The heart of the system is a PC-driven imaging workstation. The computer hardware features a variety of real-time digital image processing techniques including bias/gain control, signal averag ing, shading correction, and background subtraction. Analysis of the processed image is accomplished with software programs that are continuously upgraded and available to the customer free of charge (up to one year after the date of purchase). Software-directed image analysis features allow the user to perform tasks such as: 1) displaying the results either as dimensionless ratios or quantitatively mapped to calcium, sodium, or pH values via user-provided reference intensities; 2) dualframe registration checking via bit-plane overlay to ensure that pixels at one wavelength match those at another wavelength; and 3) interrogation of values by individual pixel, 8x8 pixel cursor, or an arbitrarily defined region of interest. In addition, image processing and coloration operations may be specified to include/exclude topological features defined by intensity area, or position. Thus, selected shapes, such as an individual cell, may be analyzed selectively. Hard-copy images may be obtained by output to an HP Laser Jet and compatible laser printers in a 16-level pay scale at 300 dots/inch. Images maybe fully annotated with text and line art. Wendy Wilson Sheridan is associate director of foundation and corporate relations, and V. Richard Sheridan is a postdoctoral associate in pharmacology, both at Fox Chase Cancer Center, Philadelphia.

Fluorescence Illumination Lights Up Living Cells

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