Detection and Quantification of Bacterial Autofluorescence at the Single-Cell Level by a Laboratory-Built High-Sensitivity Flow Cytometer

Abstract

Cellular autofluorescence can affect the sensitivity of fluorescence microscopic or flow cytometric assays by interfering with or even precluding the detection of low-level specific fluorescence. Here we developed a method to detect and quantify bacterial autofluorescence in the green region of the spectrum at the single-cell level using a laboratory-built high-sensitivity flow cytometer (HSFCM). The detection of the very weak bacterial autofluorescence was confirmed by analyzing polystyrene beads of comparable and larger size than bacteria in parallel. Dithionite reduction and air re-exposure experiments verified that the green autofluorescence mainly originates from endogenous flavins. Bacterial autofluorescence was quantified by calibrating the fluorescence intensity of nanospheres with known FITC equivalents, and autofluorescence distribution was generated by analyzing thousands of bacterial cells in 1 min. Among the eight bacterial strains tested, it was found that bacterial autofluorescence can vary from 80 to 1400 FITC equivalents per cell, depending on the bacterial species, and a relatively large cell-to-cell variation in autofluorescence intensity was observed. Quantitative measurements of bacterial autofluorescence provide a reference for the background signals that can be expected with bacteria, which is important in guiding studies of low-level gene expression and for the detection of low-abundance biological molecules in individual bacterial cells. This paper presents the first quantification of bacterial autofluorescence in FITC equivalents.

Green autofluorescence, a double edged monitoring tool for bacterial growth and activity in micro-plates

“…Their contribution to eukaryotic cellular autofluorescence started long ago to be addressed [16] and different methods to separate their fluorescence from exogenous GFP reporters are currently explored [17], [13]. In prokaryotes the focus has been mainly on internal flavin driven autofluorescence, for example for bacterial cell counting with microfluidic diagnostic devices [18] or for autofluorescence quantification with dedicated flow-cytometers [19].…”

Quantitative Comparison of the Autofluorescence of Bacteria and Polystyrene Microspheres under Violet Wavelength Excitation for Verification of Fluorescence-based Bioaerosol Detector Results

“…As the results, the microbial intensity was found to be similar to the autofluorescence intensity of some kinds of PSMs Fig. 2 as producing background noise in high-sensitivity analysis using fluorescence dye Yang et al, 2012 . We have added information about artificial particles as comparable reference particles with regard to microbial autofluorescence intensity.…”

PROTELPHIDIUM NIVEUM() AND THE TAXONOMY OF “LOWER” ELPHIDIIDS

“…As recently demonstrated (Apothéloz-Perret-Gentil et al, 2013), at least one species of foraminifera is able to emit green autofluorescence (GAF) of unknown origin. On the other hand, GAF is widely distributed among microalgae, including diatoms (Tang & Dobbs, 2007) and bacteria (Rost, 1995;Yang et al, 2012). Consequently, we do not exclude the possibility that green autofluorescent bodies in P. niveum are derived from its food.…”