We propose the use of a 405 nm laser to excite the cofactor F420. The study tested this excitation source for precise determination of abundance and autofluorescence intensity of F420 fluorescent cells in a variety of methane producing microbial communities. These include a methanogenic enrichment culture and several communities from continuously operated digesters fed with industrial grade renewable or lab-desigend substrates. We present a method including quality control steps for a reliable application in biogas reactor environments. The procedure was verified by MiSeq sequencing of flow cytometrically sorted subcommunities and T-RFLP analysis of whole samples. For more details ask to the corresponding author susann.mueller@ufz.de.