Overcoming the diffraction limit through FLIM-integrated STED nanoscopy while preserving living samples from phototoxicity

Session

Poster Session

Authors

Damien Schapman (1), Magalie Benard (1), Christophe Chamot (1), Aurelien Debonne (1, 3), Alexis Lebon (1), Hitoshi Komuro (1), Fatemeh Dubois (2), Guénaëlle Levallet (2), Ludovic Galas (1)

Affiliations

1. Univ Rouen Normandie, INSERM, CNRS, Normandie Université, HeRacLeS US51 UAR2026, PRIMACEN, F-76000 Rouen
2. Université de Caen Normandie, CNRS, Normandie Université, ISTCT UMR6030, GIP CYCERON, F-14000 Caen
3. Univ Rouen Normandie, INSERM U1245, F-76000 Rouen

Keywords

Tunneling nanotubes; live-cell imaging; sample preservation; fluorescence lifetime; FLIM; STED

Abstract text

Here, we proposed a challenging advanced light imaging strategy to reveal the dynamics of Tunneling NanoTubes (TNTs) that are now considered as a long and short distance route for cell-to-cell communication.

Fluorescence microscopies are essential to decipher physiological and pathophysiological processes. However, the preservation of living samples during imaging is a crucial key element to avoid any artefact or misinterpretation. Therefore, a challenging compromise is necessary to achieve appropriate spatial and temporal resolution and acceptable signal to noise ratio without phototoxicity. In most cases, cell imaging through wide-field fluorescence microscopy, TIRFM or spinning disk/resonant scanner confocal microscopy is sufficient to reveal cell movements and intracellular dynamics but the task becomes more complex and more difficult when the size of biological objects is less than 200 nm. To overcome the diffraction limit, super-resolution approaches including STED nanoscopy, have been developed with however the risk to damage the biological sample due to high light exposure. The aim of this work was to propose a strategy to image ultrathin, fragile and floating cell-to-cell connections so called Tunneling NanoTubes with a combination of fast FLIM and STED nanoscopy.

Pleural mesothelial H28 cells line were used as a biological model since they expressed spontaneously type 1 and 2 Tunneling NanoTubes in normal culture conditions. H28 cells were labeled with single or multiple dyes including SPY650-FastAct, Red MitoTracker, LBL-Dye M715, and Nile Red. An inverted confocal laser scanning microscope (STELLARIS 8, Leica Microsystems) equipped with a white light laser (440-790 nm), four hybrid detectors (HyD type S, X and R), 86x (NA = 1.20, water immersion, WD = 300 µm), 93x (NA = 1.30, glycerol immersion, WD = 130 µm) and 100x (NA = 1.40, oil immersion, WD = 300 µm) objectives and a conventional scanner (400 Hz, 1024x1024). Airy 1 pinhole was used. A fully fast integrated FLIM module so called FAst Lifetime CONtrast (FALCON, Leica Microsystems) and a STED module with 592-nm and 775-nm pulsed depletion lasers were used to perform nanoscopy. Laser power, irradiance and transmission data were obtained via an Argo-POWER slide (Argolight, Talence, France) which integrates an optical power meter. HyD-S, HyD-X and HyD-R sensitivities were determined with three homogeneous fluorescent solutions of fluorescein, rhodamine B and rhodamine 800. HyDs were used in photon counting mode. Mean signal intensity of the whole images (ImageJ) revealed HyD sensitivities.Bead (FluospheresTM Carboxylate-Modified Microspheres, F8795 and F8789, Thermo Fisher Scientific, Illkirch-Graffenstaden, France) imaging was used to compare objective performances through STED nanoscopy. Full Width Half Maximum (FWHM) measurement of bead fluorescence signal with ImageJ led to determination of 86x, 93x and 100x lateral resolution.

In this study, the characterization of key instrumental elements of our confocal laser scanning microscope equipped with fast FLIM and STED modules first revealed that whatever the objective, the lowest irradiance values were detected for i) red laser excitation wavelengths (WLL, constant laser power, 47 µW; 35-55 kW.cm⁻²) and ii) for 775-nm depletion laser. Second, type X and S of hybrid detectors (HyD-X, HyD-S) were particularly adapted for red/near-infrared photon counting and τ separation. Using fluorescent beads and 775-nm (60 %) STED nanoscopy, the 86x objective showed better lateral resolution (47±3 nm) than 93x (57±2 nm) and 100x (73±3 nm) objectives. Interestingly with only 20% of the 775 nm-depletion power, combination of FLIM and STED approaches together with a 86x objective allowed a significant improvement in lateral resolution (from 233 ± 10 nm to 54 ± 2 nm, *** p < 0.001). Therefore, photon-counting mode and sensitivity of HyDs together with phasor plot analysis of fluorescence lifetimes enabled the flexible and fast imaging of multi-labeled living H28 cells. In these conditions, vesicle-like structures, tubular and rounded-shape mitochondria were identified within living TNTs. Through fast-FLIM-integrated STED nanoscopy, the average speed of Nile Red positive lipid droplets in TNT1 and TNT2 were 0.76 ± 0.24 µm/min and 4.86 ± 0.6 µm/min respectively. When double-labeling of H28 cells was performed with MitoTracker Red (MTR) and SiR700-Act (for actin labeling), optimization of the configuration for fast FLIM-integrated STED nanoscopy and simultaneous acquisition with two detectors were required. The activation of a HyD-S detector and an excitation wavelength 590-nm with 1% laser power was dedicated to MTR, while activation of a HyD-X detector and an excitation wavelength 698-nm with 6% laser power was dedicated to SiR700-Act. A single 775-nm STED depletion laser (2%) was used. The SiR700-Act signal in HyD-X was denoised (STED lifetime denoising) and used for the final merged image, while the MTR signal in HyD-S was used for lifetime imaging.

The characterization of objectives, HyD detectors, light sources and associated irradiance were studied to consider the best instrumental configuration for live-TNT imaging.  Red and near-infrared bright and photostable dyes were used for low energy excitation.  Thanks to a fast FLIM module integrated to STED nanoscopy, lifetime imaging, lifetime dye unmixing, and lifetime denoising techniques were applied to perform time-lapses of tens of minutes revealing therefore structural and functional characteristics of living TNTs that were preserved from light exposure.