AxioVision Ratio

While intracellular processes are taking place it is often possible to observe changes in ionic concentration (Ca, Na, K) and pH value. These changes in concentration are studied using fluorescence-labelled indicator molecules that emit light of a defined wavelength when subjected to fluorescence excitation. Such processes can be quantified with the help of suitable imaging systems.

The Ratio module is integrated into AxioVision directly as an application therefore expanding its functionality to include ratio measurements in fluorescence images. The individual processing steps are integrated into a clearly presented dialogue offering the user simple and flexible operation: from calculating the ratio image and marking areas for measurement to displaying measurement results.

To measure ratios by means of image analysis it is first necessary to acquire the fluorescence signals using appropriate hardware and software (e.g.motorized fluorescence microscope with AxioVision and the Multichannel Fluorescence and Time Lapse modules).
In most cases two fluorescent dyes are used which absorb/emit different wavelengths and therefore serve as a measurement signal and a reference signal.

The ratio image is created as a quotient of the two fluorescence signals and displays a pseudo-colour coding of the fluorescence ratios, with warmer colours representing greater differences and colder colours smaller ones.
To balance out a varying background it is possible to perform a correction for each fluorescence channel prior to the calculation of the ratio.

In the ratio image, the fluorescence intensities of individual cells can be measured simply by outlining them using the mouse. To distinguish between different cell types you are given a free choice of the colour used for marking and of the description used for the cell.
The simultaneous display of raw data, intensity graphic and ratio image allows the marked cells and corresponding measurement values to be directly compared.