Label free imaging with harmonic generation microscopy and endogeneous fluorescence investigations for food, ecotoxicology and health issues

Session

LSA.1 - Label-free life science imaging

Authors

Dr Laurence Dubreil (2), Dr Sylvie Chevalier (1), Dr Jérôme Cachot (3), Pr Marie-Anne Colle (2), Dr Karl Rouger (2)

Affiliations

1. Oniris, Université de Nantes, CNRS, GEPEA, UMR 6144, 44000
2. PAnTher, INRAe, École Nationale Vétérinaire, Agro-alimentaire et de l'alimentation Nantes-Atlantique (Oniris), Université Bretagne Loire (UBL), 44307
3. Université de Bordeaux, CNRS, EPOC, UMR 5805, 33600

Keywords

Label free, Multiphotonic microscopy, food, ecotoxicology, health

Abstract text

Multiphoton microscopy, combining two-and three photons excited intrinsic fluorescence, second harmonic (SHG) and third harmonic generation (THG), has become an important tool for high-resolution, non_invasive imaging of biological tissues. 

SHG occurs at ordered non-centrosymetric structure. Starch grains and collagen fibers represent the major source of SHG in plant and in animal tissues, respectively. THG occurs at structural interfaces, such as local transitions of the refractive index. Therefore, THG microscopy with approximately 1200 nm excitation is a powerful tool for examining the distribution of lipid bodies in a set of unstained samples including plant seeds and intact mammalian tissue.

Here, we demonstrate the high potentialities of multiphoton microscopy with label free investigation for food, environment and health issues. Sample preparation and image acquisition protocols are described. Both harmonic signals and autofluorescence will be investigate for plant and animal tissues phenotyping with absence of exogenous labeling. 

Measurements were performed on a Nikon microscope A1RMP-HD coupled with an InsightDeepsee laser (Spectra Physics), used in the 820-1300 nm range < 120 fs pulse width. An auxiliary beam at 1040 nm was available in combination with the tunable output for dual wavelength excitation. Short emission filters were used according to excitation wavelengths in order to separate specifically fluorescence and harmonic signals.

 

A first project was related to food issues with a label free detection of starch granule in potatoe fried product for a better understanding of food structure. By using non linear microscopy and specific emission filters, we demonstrated the SHG detection from starch granule in the fried product without highly destructive staining. This label-free detection thanks to second harmonic generation investigation from starch granules in fried food is a very promising approach for microstructure investigation of a large panel of starchy food products [1].

A second project concerned environmental issues with the ingestion of microplastic (MPs) in Japanese medaka larvae and their associated toxic effects. Thanks to multiphotonic microscopy allowing higher beam penetration and 3D imaging with optical sectioning, MPs were investigated in whole larvae. They were detected in digestive tract by using optimized combination between excitation wavelength and band pass emission filters used to discriminate natural fluorescence of MPs and endogeneous fluorescence of medaka [2]. This work demonstrated the advantages of label-free detection in ecotoxicology issues, allowing to acquire direct information of the contaminants based on/using their spectral properties.

A third project was related to health issues with label-free phenotyping of skeletal muscle in two diseases corresponding respectively to glycogen storage disease type II (Pompe disease) and Duchenne Muscular Dystrophy. In Pompe disease, autophagy that is known as playing a major role in the pathogenesis was investigated thanks to a LC3-GFP transgenic Pompe mice model. Combination of clearing method and two photon fluorescence imaging allowed us to analyze skeletal muscle with 2 mm deep to assess importance of autophagy from LC3-GFP expression in the pathologic muscle. In dystrophic muscle, the progressive replacement of muscle by connective tissue (SHG imaging) and adipose tissue (THG imaging) was followed thank to harmonic microscopy [3]. Furthermore, we demonstrated that information content from SHG collected in forward and backward directions could discriminate connective tissue from dystrophic and healthy muscle. This specific signature of the collagen remodeling could provide a novel marker to document the disease course at the tissue level and to assess the potential benefit of therapeutic strategies.

References

[1] A Chouët et al, Sensors (Basel). 2019;19(9):2024.

 

[2] P Pannetier et al, Environ Int. 2020;134:105047.

 

[3] L Dubreil et al, ACS Nano. 2017;11(7):6672–6681. 

 

[4] The authors gratefully acknowledge APEX platform of the INRAe/Oniris UMR 703, Center of Excellence Nikon Nantes. MP acquisition was supported by a grant from Investissement d’Avenir (ANR-11-INBS-0011-NeurATRIS : A Translational Research Infrastructure for Biotherapies in Neurosciences) and the Région Pays de la Loire.