## 3 Results and Discussion
A comparison of the ranges of average fluorescence lifetime of the analyzed substances at excitations of 280, 340, and 460 nm is shown in Figure 6.  
Figure 6 shows that the smallest variation in average fluorescence lifetimes occurs in the case of excitation at a wavelength of 280 nm. This is understandable because proteins responsible for this excitation consist, among others, of three fluorescent amino acids, of which the fluorescence of phenylalanine can be neglected, and tryptophan and tyrosine have similar emission decay times. However, the excitation wavelength at about 280 nm is universal for all biological materials and is often used in early BWA detection devices. It should be emphasized that the analysis of fluorescence decay characteristics at this excitation wavelength should be used to detect the presence of biological agents and not to classify them. Excitation wavelengths at 340 and 460 nm are more usable for classification purposes. Lifetimes of  
Figure 5: Decay characteristics of vegetative bacteria, bacterial spores, and fungi excited at wavelength 280 nm using the stroboscopic technique. Figure 6: Ranges and frequency of occurrence of mean fluorescence times at 280, 340, and 460 nm excitations. vegetative bacteria, pollen, and spores are best separated at 340 nm excitation, while fungi are clearly distinguished at 460 nm. This is primarily due to the large variety of different fluorophores, excited at these wavelengths, contained in these materials. The averaged parameter represents the relationship between the samples more uniformly when compared to the two or three lifetime components. It appears that at each excitation wavelength, the pollens display the shortest mean lifetimes, while the fungi display the longest. It can be concluded that fluorescence lifetimes vary from group to group depending on the excitation wavelength. Those differences suggest that fluorescence decay times could potentially be used to distinguish between various groups of microorganisms. However, the lifetimes and the number of components can vary depending on the fitting procedure, which points to quite an arbitrary character of lifetime data analysis. The results can also depend on excitation and emission wavelength [26; 52]. It can be observed that pollens display shorter lifetimes than other considered groups. The first two components exhibit a lifetime of 1-2 ns at each of the excitation wavelengths. However, at 280 nm and 460 nm, the percentage contributions of the first and the second components are similar. The third component displays a short lifetime value at excitation at 280 nm, whereas at 340 and 460 nm excitation wavelengths, it was shifted to longer times around 6-7 ns. The contribution of the third lifetime component at each excitation wavelength is relatively constant and reaches about 10%. The bacterial spores are characterized by a 90% contribution (0.5-1.5 ns) for the first component at all excitation wavelengths examined. This is a significant feature as compared to other biological materials due to the absence of the third component for 340 nm and 460 nm excitation. Fungi display a relatively long lifetime at all examined excitation wavelengths, and individual results are rather diverse, especially at 340 nm and 460 nm excitation. An example of the percentage of individual fluorescence decay times for 340 nm excitation is shown in Figure 7.  
The difficulties in FD time comparison can be avoided by a different analytical approach--Principal Component Analysis (PCA). This allows results to be obtained that are reliable and independent of the operator. It has been demonstrated that clustering methods in spectroscopy are suitable for the classification of biological material. The great advantage of PCA is its ability to produce real-time data analysis based on the spectral characteristics of the sample [28; 53]. Additionally, results are independent of the curve fitting procedure. Figure 7: Fluorescence lifetimes and component contribution of bacteria, spores, fungi, and pollens excited at 340 nm.