Rapid ‘on-line’ Image Processing as a Tool in the Evaluation of Kinetic and Morphological Aspects of Receptor – induced Cell Activation

Abstract

Transmembrane signalling involves rapid and spatially well defined changes in cytosolic free Ca²⁺, [Ca²⁺]_i. Specific technologies involving image processing permit the analysis of kinetic and morphological aspects of [Ca²⁺]_i at the subcellular level with the fluorescent Ca²⁺ probe fura-2. Fluorescence excitation wavelengths (340 nm or 380 nm) are alternated in synchrony with the acquisition at video rate of images captured with an intensified CCD camera. Images are digitized, recursively filtered, divided, and displayed after calibration of the ‘ratio’ image into a numerical [Ca²⁺]_i scale. The image processor IMAGINE (Synoptics Ltd., UK) permits these operations at video rate. This produces ‘on-line’ [Ca²⁺]_i images in real time which are stored on video tapes for subsequent analysis. The present communication summarizes the rationale for the selection of our current technologies. A comparison with alternative solutions should highlight the particular advantages and drawbacks of our approach. The present text thus should serve as a help for investigators who try to assemble image processing tools for work in the receptor and cellular signalling field.