Label-free multimodal imaging of infected Galleria mellonella larvae

Abstract

Non-linear imaging modalities have enabled us to obtain unique morpho-chemical insights into the tissue architecture of various biological model organisms in a label-free manner. However, these imaging techniques have so far not been applied to analyze the Galleria mellonella infection model. This study utilizes for the first time the strength of multimodal imaging techniques to explore infection-related changes in the Galleria mellonella larvae due to massive E. faecalis bacterial infection. Multimodal imaging techniques such as fluorescent lifetime imaging (FLIM), coherent anti-Stokes Raman scattering (CARS), two-photon excited fluorescence (TPEF), and second harmonic generation (SHG) were implemented in conjunction with histological HE images to analyze infection-associated tissue damage. The changes in the larvae in response to the infection, such as melanization, vacuolization, nodule formation, and hemocyte infiltration as a defense mechanism of insects against microbial pathogens, were visualized after Enterococcus faecalis was administered. Furthermore, multimodal imaging served for the analysis of implant-associated biofilm infections by visualizing biofilm adherence on medical stainless steel and ePTFE implants within the larvae. Our results suggest that infection-related changes as well as the integrity of the tissue of G. mellonella larvae can be studied with high morphological and chemical contrast in a label-free manner.

Galleria mellonella–intracellular bacteria pathogen infection models: the ins and outs

“…In addition, label-free multimodal imaging methods may be particularly informative. These include fluorescent lifetime imaging (FLIM), coherent anti-Stokes Raman scattering (CARS), two-photon excited fluorescence (TPEF), and second harmonic generation (SHG), all used in conjunction with histological images to analyse G. mellonella infection-associated tissue damage (Quansah et al 2022 ). The methods were also applicable to bacteria growing as a biofilm, a mode of growth that may be important for M. tuberculosis survival in vivo , including caseous necrosis and cavity formation in lung tissue (Esteban and García-Coca 2018 ).…”

Studies of Streptococcus anginosus Virulence in Dictyostelium discoideum and Galleria mellonella Models

“…They include proteins homologous to the complement system, antibacterial peptides, and melanin ( 31 , 32 ). The larvae can be used for subcutaneous injection, oral administration, and in vivo imaging ( 33 , 34 ), for monitoring intracellular gene and protein expression ( 35 – 37 ), to detect immune responses ( 38 ), and to test antimicrobial drugs ( 39 , 40 ). The use of this surrogate infectious model does not require special facilities; its short time and cost efficiency are significant benefits, and it does not raise ethical concerns.…”