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Introduction Material and Methods Experimental Results and Discussion Experimental Results and Discussion (cont.) Experimental Results and Discussion (cont.) Conclusion References

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The experimental set up, designed to measure photons emitted from biological systems, consists in general of a steel dark chamber where the samples to be analysed can be maintained at a constant temperature. The radiation emitted from the sample is detected by a low-noise photomultiplier working in single photon counting mode and having a spectral sensitivity ranging from 200_nm to 850_nm. In order to decrease the dark current the photomultiplier is cooled down to -30°C . Due to the very low intensity of the signal it is necessary to increase as much as possible the solid angle of measurements but, because it is necessary to put in shutters and optical filters and to heat insulate the photomultiplier from the surrounding, in a standard experimental set up the solid angle of measurements is of the order of .1 sr.

Several kinds of light emitting devices ranging from high intensity LEDs to halogen lamps can be used as sources of exciting radiation but for all these devices is necessary to ensure that the emission power is stable in time and that the area covered by the light is illuminated with uniform intensity.

Moreover some care is necessary because with rather long periods of illumination the parameters describing time decay could not be connected only to the state of the system but also to the temporal duration of the illumination. In order to avoid such undesirable effect a source which presents extremely short times of illumination (about 3ms) was used for about all the experiments.

Measurements consist of illuminating a biological sample and in counting the number of photons re-emitted from the sample after the light source had been switched off.

During the illumination a light shutter, above which the photomultiplier is fastened, is closed in order to prevent the dimpling of the photomultiplier. After the light source was switched off the shutter is opened by an electromagnetic actuator. Due to this time lag of the experimental set-up , the photon counting starts some tens of ms after the source is switched-off.

The counting of photons emitted by the sample, after each illumination, is stored by a channel scaler using a dwelling time chosen in order to measure the decay dynamics in the best way .

Due to the low level of the signal a spectral analysis of the emitted photons could be performed only by using broadband filters corresponding to rather extended spectral intervals (of the order of tens of nm).

In general it is necessary between two successive illuminations to wait for a time interval long enough to enable the emission from the biological sample to return to, within the experimental error margin, the value prior to the perturbation. This time is at least ten times greater than the time necessary for the signal to reach the background value.

In order to reduce the environmental influence and to avoid residual luminescence from the materials, it is necessary to maintain each sample, i.e. the biological sample located in its container ( generally a plastic or quartz cuvette) , in the dark room several hours before starting the measurements.

Because it is not possible to neglect the background emission one has to measure the yield of photons emitted from the container of the sample filled only with the culture medium (if it is present) in the same experimental conditions for each set of measurements, in order to obtain the correct background values that will be subtracted from the measurements taken from each sample.

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