Tcs sp8x laser

Confocal optical sectioning is performed with on a Leica TCS-SP8X laser-scanning confocal microscope (Leica Microsystems, Mannheim, Germany) equipped with a white light laser (WLL) source and a 405nm diode laser. Sequential confocal images are acquired using a HC PLAPO 63x oil-immersion objective (1.40 numerical aperture, Leica Microsystems). Z-reconstructions of serial single optical sections are acquired every 3 min (for SiR-Actin probe) or 5 min (for SiR-Tubulin probe), and carried out with a 512x512 format, scan speed of 400Hz, a pixel size of 0,3 μm, and z-step size of 0,5 μm. Lasers’ power, beam splitters, filter settings, pinhole diameters and scan mode are the same for all examined samples of each staining. Time-lapse microscopy was performed with a stage incubator (OkoLab, Naples, Italy) allowing to maintain stable conditions of temperature, CO₂ and humidity during live-cell imaging.
Z-reconstructions are imported into Imaris (Bitplane, Zurich, Switzerland) software to obtain their three-dimensional (3D) surface rendering. To improve contrast and resolution of confocal raw images, deconvolution analysis (3D Deconvolution software, Leica Microsystems) is applied to Z stacks before 3D reconstruction. The reconstructed images were assembled in Adobe Photoshop CS6 software (Adobe Systems Inc., San Jose, CA).

Confocal microscopy was performed on a Leica TCS-SP8X laser-scanning confocal microscope (Leica Microsystems, Mannheim, Germany) equipped with white light laser (WLL) source, 405nm diode laser, SuperZ Galvo stage, 3 Internal Spectral Detector Channels (PMT) and 2 Internal Spectral Detector Channels (HyD) GaAsP. Sequential confocal images were acquired using a HC PLAPO 63x oil immersion objective (1.40 numerical aperture, Leica Microsystems) with a 1024×1024 format, scan speed 600Hz, an electronic zoom at 1.3 corresponding to 136 nm/pixel, and z-step size of 0.3 mm. Z-reconstructions (60 stacks) were imported into LAS AF 3D Analysis (Leica Microsystems) software to obtain their three-dimensional (3D) surface rendering.