Authors:
Cédric Delmon
1
;
Erwan Ferrandon
2
;
Emilie Chouzenoux
3
;
Audrey Prorot
1
;
Sophie Alain
4
and
Claire Lefort
2
Affiliations:
1
Peirene, GRESE (EA 4330), University of Limoges, Limoges, France
;
2
XLIM Research Institute, UMR CNRS 7252, University of Limoges, Limoges, France
;
3
Center for Visual Computing, CentraleSupélec, Inria Saclay, Université Paris-Saclay, Gif-sur-Yvette, France
;
4
RESINFIT, UMR INSERM 1092, University of Limoges, Bacteriology-Virology-Hygiene Department, University Hospital Center, France
Keyword(s):
Multiphoton Microscopy, Bacterial Imaging, Spectral Characterization, Two-photon Excitation, Fluorescence Emission, Metabolic Indicators, Point-Spread-Function, Computational Restoration, Block Distributed Majorize-minimize Memory Gradient.
Abstract:
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-photo
n 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.
(More)