## 2 Materials and Methods
### Registration of Ultra-Weak Photonic Emission  
The research was carried out using a proprietary measurement system consisting of elements that enabled photon registration based on the electromagnetic radiation emitted from living organisms. A photomultiplier HAMAMATSU type R4220 (Hamamatsu Photonics KK, Hamamatsu, Japan) was used for registration and photon detector. The light-emitting sample is placed in the measuring chamber. The chamber is thermally stabilized depending on the type of tests performed. The light-emitting sample is placed in a lightproof measuring chamber. The lightproof chamber is equipped with a shield system for periodically obscuring photon registration Consequently, the measurement and emission noise value are measured alternately. This is especially important when the intensity of the emitted radiation is residual and the recorded signal is not much different from the measurement noise. The single photon counting method was used to determine the ultra-low photon emission. The time interval of the single photon counting operation was set experimentally in each case, depending on the material tested (egg shell, egg white, egg yolk). The minimum length of the sample residence interval in the lightproof chamber was assumed to be the correct time when the difference in the number of photons counted between two immediately adjacent 1 min intervals was less than 10%. The result of the measurement of ultra-low photonic emission is the absolute difference between the number of photons registered by the photomultiplier in the lightproof chamber with the material and the number of photons registered by the photomultiplier in this chamber without material, according to L = A - B (photons), where: L = number of photons emitted by the tested sample, A = number of photons emitted by a sample placed in a lightproof chamber, B = number of indications (photons) generated by an empty lightproof chamber. Calibration of the sensor was carried out each time on the day of the measurements, and consisted of determining the ratio of the system response to the standard dose of radiation according to Equation (2). This results from the test procedure for which the department is accredited by the research laboratory of the Polish Center for Accreditation no. AB 169, and it is necessary to ensure an appropriate budget for the uncertainty of the measurement results:  
\[K=\frac{A_{o}-B}{D}, \tag{2}\]  
where:  
\(K\)--calibration coefficient--if the calibration factor is in the range of (0.8-1), the measuring system is considered to be efficient and ready for measurement. \(A_{o}\)--number of photons counted in the interval of 500 s, with reference forcing,  
\(B\)--number of photons registered in an empty lightproof chamber,  
\(D\)--known dose used for calibration (400 photons). After starting the measuring system in the first phase with a time interval of 120 s, stabilization of the system occurred to prevent interference arising from temporary destabilization of standard conditions. After the initial phase, the main (measuring) phase ensued, with a time interval of 500 s. The frequency of the result recording was 4 Hz, i.e., each recorded result was the sum of photons counted within 0.25 s. These parameters were determined as mentioned above by preliminary experiments, but also included the minimum exposure time necessary for observations to lead to statistically significant test results. The main phase was followed by the final phase of the measurement, in which a stop occurs, but not by the operation of the measuring sequence. The whole measurement process was monitored in real time by our original application made in the LabView program. Eggs from cage (industrial) and free-range (traditional) housed hens were analyzed. Due to the different mass of the analyzed eggs, the results (expressed as a standardized number of photons) were converted to an egg weight of 60 g. An analysis was made of photons of 60 eggs from each type of culture in duplicate--a total of 240 samples for each part of the egg.