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Data Acquisition and Analysis

Loyola researchers are using fluorescence spectroscopy to directly monitor mitochondrial NADH and, with the aid of calcium sensitive fluorescent dyes, cytosolic and mitochondrial calcium in isolated rat cardiac muscles (trabeculae). After dissection from the heart ventricular wall, the muscle is mounted in a muscle chamber and superfused with a substrate rich solution (containing glucose and pyruvate). Increased work is obtained by increased pacing frequency (also causes increased calcium) or stretching the muscle (does not alter calcium). Muscle calcium and NADH is measured by 350 nm excitation light and detecting the emitted fluorescence at 385, 456 and 590 nm,using instrumentation designed and built by Dr. Brandes, while work is estimated from the resulting Average Force. Fluorescence spectroscopy produces a huge amount of data upon which routine analysis must be performed. In the past, Loyola researchers had to spend a considerable portion of their time manipulating this data using software packages that were less than completely intuitive. As a result, they spent more time analyzing data than performing experiments. Recently, they were able to substantially reduce the time required to analyzing data by switching to a Windows-based data analysis and technical graphics software package called Origin from OriginLab Corporation, (Northampton, MA). The Origin user interface follows Windows conventions and is based upon the procedures and terminology of scientific researchers, making it extremely intuitive to use. At the same time, Origin provides all of the analysis tools required by Loyola researchers, particularly including a wide range of nonlinear curve fitting tools and extensive multivariate regression analysis capabilities. Just as important, Origin includes an extremely powerful programming language, called LabTalk(tm) that provides access to virtually every function in the program. Using LabTalk, Loyola researchers developed scripts that automated all of their common data analysis routines. These scripts can be accessed simply by pressing a button on the screen. Origin scripts were developed to process the raw fluorescence and force data to obtain calibrated calcium, NADH and Average Force traces, while other scripts were developed to extract information from the processed data. The time needed to process the raw data and to obtain scientifically valuable information could thus be significantly reduced. The first automatic procedure consisted of reading the raw data from disk, subtracting background fluorescence, calculate ratios between different channels, calculating Average Force using Origin's software-based digital filtering capabilities, calibrating the fluorescence and force signals, create figures and printing them, and finally storing the processed data on disk. The second automatic procedure consisted of extracting information from the calculated NADH, calcium and force data and storing it to disk.

 

 

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