Cytochrome-c based Resonance Raman Microscopy: analyzing bacterial phylogeny in vivo

Cytochromes c (cyt-c) are ubiquitous electron transfer proteins found in the majority of life forms on earth. Any change to the heme moiety, a porphyrin embedded in the protein matrix, results in total loss of protein function. However, the protein matrix supporting the heme is highly susceptible to evolutionary change. Confocal Resonance Raman Microscopy tuned for the porphyrin lattice vibrations of the heme moiety can be used to record the cyt-c Raman spectrum of individual bacterial cells in vivo and in situ. The Raman spectra are then categorized into clusters based on their spectral similarity, determining the similarity of the cytochrome-content of the respective cells and ultimately, how evolutionary close the analyzed cells are to each other. We present a proof of concept determining the relative relatedness of four bacterial strains (3 species of Nitrosomonas and a mutant Rhodobacter) based on the similarity of their cyt-c resonant Raman spectra recorded in vivo from individual cells in planktonic culture.