Multiphoton Microscopy for Bacterial Imaging: A Label-free Solution Resting on Endogenous Two-photon Fluorescence

We demonstrate the interest of multiphoton microscopy (MPM) for imaging bacteria without any labelling process. Six families of bacteria are tested: Escherichia coli, Staphylococcus epidermidis, Proteus vulgaris, Pseudomonas fluorescens, Bacillus subtilis and Clostridium perfringens. For each of these bacteria, the image of a cell is recorded through a multiphoton microscope thus revealing the 3D shape of these bacteria. For the first time, the images of such bacteria are recorded without any labelling solution. A protocol of controlling the image produced is led thanks to a standard staining protocol with carboxy fluorescein diacetate (CFDA) for E. Coli and Staphylococcus epidermidis. Similar object shapes with or without labelling are produced, thus validating the label-free images generated by MPM. Then, the two-photon excitation spectra are measured for each of these bacteriaand the emission spectra delivered by E. Coli and Bacillus subtilis are shown. The origin of the two-photon fluorescence (TPF) emission of the bacteria thanks to the nonlinear imaging solution is discussed regarding to the TPF excitation and emission spectra of metabolic indicators.