For these reasons, considerable efforts have been made to develop methods to deal with these nuisances, which are referred to as background fluorescence, noise, or spectral crosstalk 1, 2. In addition, because cellular extracts are frequently components of the growth media, growth media are also commonly autofluorescent. Correction of this “contaminating” AF is problematic because it is frequently unevenly distributed within and between cells. For example, AF precludes the detection of weak signals from the fluorescent reporters for low-abundance proteins. For this reason, AF frequently overlaps with the spectrum of exogenous fluorophores used for research purposes, and therefore interferes with the fluorescent microscopy and cytometric analyses. For example, flavins, NAD, and lipofuscin emit green, blue, and orange light respectively when excited at appropriate wavelengths. Cellular AF spectra encompass most of the spectral range because different endogenous fluorophores emit at different wavelengths of the electromagnetic spectrum. An observed increase in AF under stress is an evolutionary conserved phenomenon as it occurs not only in cells from different bacterial species, but also in yeast and human cells.Īll prokaryotic and eukaryotic cells exhibit an intrinsic natural fluorescence (autofluorescence AF) due to the presence of different fluorescent cellular structural components and metabolites, such as flavins, nicotinamide-adenine dinucleotide (NAD), aromatic amino acids, lipofuscins, advanced glycation end products, and collagen 1, 2. An increased expression of genes encoding diverse flavoproteins which are involved in energy production and ROS detoxification, indicates a cellular strategy to cope with severe stresses. Excitation and emission spectra and increased expression of the genes from the flavin biosynthesis pathway, strongly suggested that flavins are major contributors to the increased AF. We observed that bactericidal treatments increased green cellular AF, and that de novo protein synthesis was required for the observed AF increase. We examined how exposure to the different stressors changes the AF of Escherichia coli cells. Therefore, cellular autofluorescence (AF) is expected to vary with the metabolic states of cells. Prokaryotic and eukaryotic cells exhibit an intrinsic natural fluorescence due to the presence of fluorescent cellular structural components and metabolites.