Tcs sp8 system

Confocal laser scanning microscopy was conducted using a Leica TCS SP8 system (Leica Microsystems, Wetzlar, Germany). Cells were seeded in ibitreat dishes (ibidi, Munich, Germany) and grown for 72 h. For immunofluorescence staining, cells were fixed using 4% PBS-buffered formaldehyde and subsequently stained with primary and secondary antibodies using standard protocols. Counterstaining of filamentous actin and nuclei was carried out using phalloidin-TRITC and DAPI, respectively. Excitation of fluorophores was achieved using integrated lasers emitting at 405 nm (for DAPI), 488 nm (for secondary antibodies labeled with Alexa488), 552 nm (for phalloidin-TRITC) and 638 nm (for secondary antibodies labeled with Alexa647). For AO live cell imaging, excitation of the dye was performed using the 488 nm laser. Neutral compartments of the cell were detected by collecting emitted light at 493–547 nm (designated as “AO neutral”), whereas acidic compartments were detected by collecting emitted light at 575–739 nm (designated as “AO acidic”), respectively. Fluorescence was detected using a HP CL APO 63 × /1.40 OIL CS2 oil immersion objective (Leica Microsystems) and a pinhole setting of 1 Airy unit.

For confocal microscopy the TCS SP8 system in combination with the Leica Application Suite X (LasX) software were used (Leica Microsystems, Wetzlar, Germany). Cy3 was excited at 552 nm, eYFP at 488 nm and DAPI at 405 nm. Alexa Fluor 633 and 647 were excited at 638 nm.
Fluorescence emission was detected in the range of 493–548 nm for eYFP, 551–596 nm for Cy3, 410–460 nm for DAPI and 643–784 nm for Alexa Fluor 633 and 647. The fluorescence dyes were sequentially excited.

Induced Pluripotent Stem Cells-MNs were seeded on coverslips in a 24-well plate, cultured until differentiation, and infected as specified above. At 48 hpi, cells were fixed in PBS containing 4% PFA at RT for 10 min, followed by permeabilization with 0,1% TritonX-100 in PBS for 10 min. Cells were treated with 1% BSA in PBS for blocking at RT for 1 h, and incubated at 4°C overnight with specific primary antibodies. The following primary antibodies were used: anti-beta III Tubulin (1:500, Abcam, Cambridge, UK), anti-SMI-312 (1:1000, Covance, Princetown, NJ, USA) and anti-ChAT (1:200, Chemicon) to assess iPSC-MN differentiation; anti-ACE2 (1:200, Prodotti Gianni), anti-CD147 (1:100, Thermo Fisher Scientific) anti-NRP1 (1:100, Thermo Fisher Scientific) and anti-N Nucleocapsid SARS-CoV-2 (1:1000, BEI Resources) to assess SARS-CoV-2 receptors and infection. Coverslips were then stained with secondary antibodies (Alexa Fluor 488 or 647, 1:500, Abcam) for 45 min at RT and mounted using a medium containing DAPI (Enzo Life Sciences, Milan, Italy). Confocal images were acquired on a TCS SP8 System equipped with a DMi8 inverted microscope and a HC PL APO 40 × /1.30 Oil CS2 (Leica Microsystems, Wetzlar, Germany) at a resolution of 1024 × 1024 pixels (single stack).

Using published protocols [106 (link),108 (link)], MCF7 or U373 cells cultured in 120-mm coverslips were fixed in 4% paraformaldehyde in 0.1 M PB, pH 7.4, for 10 min. Cells were pre-incubated for 1 h min with 5% of normal goat serum (Life Technologies, Monza, Italy) in 0.1 M PB (pH 7.4) containing 0.1% Triton X-100, before overnight incubation with the rabbit monoclonal anti-cleaved caspase 7 (Cell Signaling Technology, Danvers, MA, USA). In double-label immunofluorescence experiments, the mouse monoclonal anti-cytochrome c primary antibody (Cell Signaling Technology) was used in conjunction with the rabbit monoclonal primary antibody directed to COX IV (Cell Signaling Technology). For fluorescence detection, coverslips were stained with the appropriate Alexa Fluor secondary antibodies (Life Technologies) and mounted on glass slides in a ProLong Gold Antifade Mountant (Life Technologies). DAPI and/or fluorescein phalloidin (cytoskeleton detection) staining was also used. Cells were analysed with a DMI4000 B automated inverted microscope equipped with a DCF310 digital camera (Leica Microsystems, Wetzlar, Germany). When indicated, confocal imaging was performed with a TCS SP8 System (Leica Microsystems). Image acquisitions were controlled by the Leica Application Suite X.

FLIM was performed on cells growing on glass µ-dishes (ibidi). In the case of fixed samples, no mounting medium was used, and FLIM was performed in PBS. Fluorescence lifetime images were acquired on a Leica TCS SP8 system attached to a Leica DMi8 inverted microscope (Leica Microsystems, Wetzlar, Germany). Excitation was provided by a white light laser with a repetition rate of 20 MHz and an acousto-optical beam splitter (AOBS) selected an excitation wavelength of 488 nm. Excitation continued for 75 s/FLIM measurement/focal plane. Images were acquired using a 63×1.4 NA oil immersion objective. Fluorescence of the H2B-GFP was detected using a hybrid detector operating in photon counting mode over an emission range of 495–530 nm. A notch filter centred on 488 nm minimised any laser scatter into the detector. Time-resolved data were acquired through use of a PicoHarp 300 TCSPC module (PicoQuant, Berlin, Germany) controlled through SymPhoTime64 software (PicoQuant). FLIM Images were acquired with 512×512 pixels and 4096 time bins. For live-cell experiments, the system was maintained at 37°C/5% CO₂. In 3D experiments, FLIM measurements were taken at a minimum of eight focal planes, 1 µm apart.

After the incubation with the secondary antibodies, and following 3 × 5 min washes in PBS, CaLu-3 cells were incubated for 15 min at RT in the dark with a solution of 0.05% w/v Thioflavin-S (T1892, Merck-Sigma, Milan, Italy), which was freshly prepared in 50% ethanol/water and 0.22 μm filtered. Cells were washed twice with 50% ethanol for 10 min each, and then washed once with 80% ethanol for 20 min. Eventually, cells were washed in PBS, briefly rinsed with water, and coverslips were mounted on Superfrost glass slides using a mounting medium with DAPI (Enzo Life Sciences, Milan, Italy). Confocal images were acquired on a TCS SP8 System equipped with a DMi8 inverted microscope and a HC PL APO 40 × /1.30 Oil CS2 (Leica Microsystems, Wetzlar, Germany) at a resolution of 1024 × 1024 pixels.

Images of the coarse emulsion O/W, the O/W carotenoid microemulsions, and the corresponding O/W microemulsions during in vitro gastrointestinal digestion were obtained using an optical microscope Axioskop (Carl Zeiss, Germany) coupled to a Leica DMC 6200 pixel shift camera (Leica Microsystems, Germany), using a Zeiss Plan-Neofluar lens with a 100× objective and the addition of an immersion oil drop. Samples were observed using an open condenser with level 4 illumination, and no color filters were used. The color was manually adjusted to show real-time colors using Leica LAS v4.13 Application Suite Software.
Microstructural analysis was also performed using confocal laser scanning microscopy (CLSM). Neutral red solution (50 μg/mL in ethanol, Sigma, Deisenhofen, Germany) was used to dye the lipophilic phase of the emulsions. The excitation and emission peaks of the dye were at 515 nm and 585 nm, respectively. This analysis was achieved using a confocal multispectral TCS SP8 system (Leica Microsystems, Mannheim, Germany) at 40× with a Zeiss Plan-Neofluar lens.