title: Simultaneous cathodoluminescence and electron microscopy cytometry of cellular vesicles labeled with fluorescent nanodiamonds.
abstract: Light and Transmission Electron Microscopies (LM and TEM) hold potential in bioimaging owing to the advantages of fast imaging of multiple cells with LM and ultrastructure resolution offered by TEM. Integrated or correlated LM and TEM are the current approaches to combine the advantages of both techniques. Here we propose an alternative in which the electron beam of a scanning TEM (STEM) is used to excite concomitantly the luminescence of nanoparticle labels (a process known as cathodoluminescence, CL), and image the cell ultrastructure. This CL-STEM imaging allows obtaining luminescence spectra and imaging ultrastructure simultaneously. We present a proof of principle experiment, showing the potential of this technique in image cytometry of cell vesicular components. To label the vesicles we used fluorescent diamond nanocrystals (nanodiamonds, NDs) of size ≈150 nm coated with different cationic polymers, known to trigger different internalization pathways. Each polymer was associated with a type of ND with a different emission spectrum. With CL-STEM, for each individual vesicle, we were able to measure (i) their size with nanometric resolution, (ii) their content in different ND labels, and realize intracellular component cytometry. In contrast to the recently reported organelle flow cytometry technique that requires cell sonication, CL-STEM-based image cytometry preserves the cell integrity and provides a much higher resolution in size. Although this novel approach is still limited by a low throughput, the automatization of data acquisition and image analysis, combined with improved intracellular targeting, should facilitate applications in cell biology at the subcellular level.
authors.affiliation: Laboratoire de Physique des Solides, CNRS, Univ. Paris-Sud, Université Paris-Saclay, 91405 Orsay, France. mathieu.kociak@u-psud.fr.
authors.forename: Sounderya
authors.initials: S
authors.lastname: Nagarajan
authors.affiliation: Signalisations, Noyaux et Innovations en Cancérologie, UMR 8126 CNRS, Univ. Paris-Sud, Université Paris-Saclay, Gustave Roussy, 94805 Villejuif, France.
authors.forename: Catherine
authors.initials: C
authors.lastname: Pioche-Durieu
authors.affiliation: Laboratoire de Physique des Solides, CNRS, Univ. Paris-Sud, Université Paris-Saclay, 91405 Orsay, France. mathieu.kociak@u-psud.fr.
authors.forename: Luiz H G
authors.initials: LH
authors.lastname: Tizei
authors.affiliation: Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei 106, Taiwan.
authors.forename: Chia-Yi
authors.initials: CY
authors.lastname: Fang
authors.affiliation: Laboratoire de Vectorologie et Thérapeutiques Anticancéreuses, UMR8203, CNRS, Univ. Paris-Sud, Gustave Roussy, Université Paris-Saclay, 94805 Villejuif, France.
authors.forename: Jean-Rémi
authors.initials: JR
authors.lastname: Bertrand
authors.affiliation: Signalisations, Noyaux et Innovations en Cancérologie, UMR 8126 CNRS, Univ. Paris-Sud, Université Paris-Saclay, Gustave Roussy, 94805 Villejuif, France.
authors.forename: Eric
authors.initials: E
authors.lastname: Le Cam
authors.affiliation: Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei 106, Taiwan.
authors.forename: Huan-Cheng
authors.initials: HC
authors.lastname: Chang
authors.affiliation: Laboratoire Aimé Cotton, CNRS, Univ. Paris-Sud, ENS Cachan, Université Paris-Saclay, 91405 Orsay, France. francois.treussart@ens-cachan.fr.
authors.forename: François
authors.initials: F
authors.lastname: Treussart
authors.affiliation: Laboratoire de Physique des Solides, CNRS, Univ. Paris-Sud, Université Paris-Saclay, 91405 Orsay, France. mathieu.kociak@u-psud.fr.
authors.forename: Mathieu
authors.initials: M
authors.lastname: Kociak