Mark Wefers Bettink

A monitor for Cellular Oxygen METabolism (COMET): monitoring tissue oxygenation at the mitochondrial level 3 47 2.2 Signal analysis Oxygen however is heterogeneously distributed in tissues in vivo . Previous studies have shown that this also applies for mitoPO 2 [6, 7, 13]. Delayed fluorescence from a heterogeneous system does not decay mono-exponentially, but the signal contains a lifetime distribution. Fitting equation 1 to a distribution of lifetimes generally leads to an underestimation of the mean PO 2 in the measuring volume [20]. A much better estimation of the mean PO 2 can be found by alternatively fitting a distribution of quencher concentration to the delayed fluorescence signal. The fitting function for a simple rectangular distribution with a mean mitoPO 2 Q m and a mitoPO 2 range from Q m - δ till Q m + δ is [21]: (2) where Y R (t) is the normalized delayed fluorescence data, < mitoPO 2 > is the mean mitoPO 2 within the sample volume and t is the factor time. Fitting of equation 2 is fast and very robust when applied to weak delayed fluorescence signals and noisy real world signals. In a previous analysis we have shown that fitting equation 2 allows for reliable retrieval of mitoPO 2 values from data with signal-to-noise ratios (SNR) as low as 10 [5]. For time-domain delayed luminescence measurements SNR is defined as the maximum amplitude of the delayed fluorescence divided by the maximum amplitude of the noise. Generally a SNR above 20 is well achievable and the noise-induced error in the measurement remains below 2%. For analysis of the delayed fluorescence signals COMET uses equation 2 to calculate mean mitoPO 2 in the measuring volume under the probe. The absolute value for mitoPO 2 is directly displayed on the screen without further processing. COMET also evaluates the signal quality, which is calculated from the SNR value; an increase of 1 in SNR is approximately 1% in signal quality up till a SNR of 50. Beyond a SNR 50 the increase in signal quality percentage will flatten out. As long as SNR is within an acceptable range, a SNR greater than 5, COMET will show a percentage and a calculated mitoPO 2 . If COMET cannot detect a signal, or SNR is too low, less than or equal to 5, the used version of the firmware makes COMET to display “no signal found” and to provide the unrealistic value of “999”.