CC BY
18LD-I-8
Label-based and label-free optical nanoscopy of pathogenic bacterial species
M. Lucidi1, S.G. Stanciu2, D.E. Tranca2, R. Hristu2, A.M. Holban3, L. Nchele1, G.A. Stanciu2, G. Cincotti1
University Roma Tre, Engineering, Rome, Italy
2University Politehnica of Bucharest, Center for Microscopy, Microanalysis and Information Processing, Bucharest, Romania
3University of Bucharest, Faculty of Biology, Microbiology and Immunology Department, Bucharest, Romania
Nanoscopy techniques can overcome the Abbe's diffraction limit, which is about half the wavelength of the excitation light, and are capable to achieve resolutions of less than 50 nm. Stimulated Emission Depletion (STED) microscopy can increase the optical resolution of a conventional confocal microscope with up to one order of magnitude, by switching off the fluorescence of dye molecules positioned in the outer regions of the excitation area with an intense doughnut shaped laser beam that depletes the electrons from the excited levels [1]. In our work, we stained different pathogenic bacterial species using the Abberior® STAR RED NHS, which has been previously utilized in STED imaging of eukaryotic cells. To our knowledge, this is the first experimental demonstration on prokaryotes' staining with this dye, and the obtained results suggest that Abberior® STAR RED NHS is able to label the membranes of both Gram-positive and Gram-negative bacteria.
We also characterized the same bacterial species with scattering-type Scanning Near-field Optical Microscopy (s-SNOM), a label-free technique for optical nanoscale imaging that we used in combination with Atomic Force Microscopy (AFM) to place the optical information into a topographic context, which is relevant for the viability of the imaged organisms. s-SNOM relies on the fact that the interaction between the enhanced near-field at the tip apex, which results upon its illumination with a focused laser beam, and the sample volume underneath modifies both the amplitude and the phase of the scattered light [2]. Besides qualitative studies, s-SNOM was recently used to determine at nanoscale resolution the refractive index (RI) of human erythrocytes [3]. For the first time, we use the same approach to determine the RI of both commensal and pathogen bacteria, which is useful for understanding in detail their optical properties and morphology.
References
[1] G. Vicidomini, P. Bianchini, A. Diaspro, STED super-resolved microscopy, Nat Methods, vol.15, pp. 173-182, (2018).
[2] F. Keilmann, R. Hillenbrand, Near-field microscopy by elastic light scattering from a tip, Philos Trans A Math Phys Eng Sci, vol. 362, pp. 787-805, (2004).
[3] D.E. Tranca, S.G. Stanciu, R. Hristu, B.M. Witgen BM, G.A. Stanciu, Nanoscale mapping of refractive index by using scattering-type scanning near-field optical microscopy, Nanomedicine, vol.1, pp. 47-50, (2018).
