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3.3. Microbial mapping The intensity of resonance Raman spectra of Cyt c allows for integration times below one second. Microbial mapping of an area of 50×50 μm2 with a resolution of 500 nm/pixel (10，000 single spectra) can be perform... 3.3. Microbial mapping The intensity of resonance Raman spectra of Cyt c allows for integration times below one second. Microbial mapping of an area of 50×50 μm2 with a resolution of 500 nm/pixel (10，000 single spectra) can be performed in less than 3 h. A further reduction of the measuring duration by a factor of 10 can be achieved by utilisation of an EMCCD camera (Coates et al.， 2004). Microbial mapping was done on granules from two different sequencing batch reactors (SBR) (Gaul et al.， 2006). The SBRs are used to analyse control parameters for the anaerobic ammonium oxidation. Fig. 7 shows two different microbial colonies in the same granulum. A graphical analysis of the spectroscopic data at 750 cm-1 (dominant resonant Raman band of Cyt c) and 2900 cm-1 (CH stretching mode) differentiated the two colonies. One colony shows stronger Raman signals at 750 cm-1 (blue frame). Whereas the other microorganisms have more amount of methyl or methylene groups (yellow frame). After the graphical analysis above， an average spectrum was constructed for each colony. The resulting averaged spectra were transferred to our Raman database of wastewater bacteria created by OPUS. The software recognized the microorganisms Fig. 5. Time series of N. eutropha Nm 57 which is captured by optical tweezers. Time difference between spectra: 1 s; Laser power: 9 mW. The bleaching effect due to photo-dissociation caused by laser radiation is easy to see. Fig. 6. The spectral heterogeneity of hierarchical clusteringis strongly dependent to the integration time. Bacteria from the same strain are only groupedtogether if the exposure time was the same. 246 R. P?tzold et al. / Journal of Microbiological Methods 72 (2008) 241–248 in the left colony as anammox bacteria， those in the upper right corner as Nitrosomonas.