Lsm 510 meta module

To evaluate sphere formation, cell-cell-contacts inside the cell agglomeration and cell performance of the outgrown cells, five-day-old pulp spheres were stained with actin, focal adhesion contacts (FAC) and 4′,6-diamidino-2-phenylindole (DAPI, Sigma) for confocal laser scanning microscopy (CLSM).
After washing and fixing, the cells were permeabilized with 0.2% Triton-X-100 in PBS and blocked with 1% bovine serum albumin (BSA, Sigma) for 30 min. FAC were stained with AlexaFluor 488-Phalloidin (Invitrogen), cytoskeletal actin was stained with AlexaFluor 546 and cell nuclei were stained with DAPI.
Microscopy was carried out by the use of an upright Axioscop 2 FS mot equipped with a LSM 510 META module (Zeiss, Jena, Germany) controlling an argon-ion (Ar+) laser, helium-neon (HeNe) laser and NIR-femtosecond titanium-sapphire laser for 2-photon excitation (Coherent Mira 900 F). The excitation of AlexaFluor 488 was carried out at 488 nm (Ar + laser) and the excitation of AlexaFluor 546 at 546 nm (HeNe laser). The NIR-fs-laser was used for the excitation of DAPI at 750 nm (2 photon excitation) and fluorescence was recorded at 461 nm.

CLSM was used for the visualization of NPs in gels. For this purpose, freeze-dried samples were swollen for up to 30 min in PBS and measured on an upright Axioscop 2 FS mot microscope equipped with a LSM 510 META module (Zeiss, Jena, Germany) using an argon+ laser for excitation of C6 at 488 nm.

Confocal images were captured using a confocal laser scanning microscope (Carl Zeiss, Jena, Germany), the LSM 510 META module, and 20× lenses. Caspase‐3 and caspase‐9 fluorescence was excited using a 488‐nm laser and emitted at 570 nm. Caspase‐1 fluorescence was excited using a 633‐nm laser and emitted at 660 nm. All images were equally processed with contrast enhancement. Images were processed using the Start LSM Image Browser.