He ne lasers

NTHi strains were grown on chocolate agar for 16 h, in the absence or presence of thymidine, and a colony was aseptically spread with an inoculation loop over a drop of distilled water on a microscopy slide. Samples were air-dried and stained for 15 min in the darkness with the cell-permeable fluorescent nucleic acid stain SYTO 9 (Life Technologies), following the manufacturer's instructions. Samples were washed twice with distilled water and fluorescence was observed by confocal laser microscopy. Images were acquired using a Leica TCS-SL filter-free spectral confocal laser-scanning microscope (Leica Microsystems) equipped with a 488 nm argon laser, 543 nm and 633 nm He/Ne lasers (Centres Científics i Tecnològics-Campus de Bellvitge, Universitat de Barcelona, Spain) using a 63× magnification oil immersion objective (1.4 numerical aperture), and an image resolution of 1024 × 1024 pixels. Images were acquired randomly and analyzed using the Leica Confocal Software 2.5 (Leica Microsystems).

Overnight bacterial cultures were used to inoculate an eight-well chambered cover glass for confocal image analysis (Ibidi GmbH) to a final OD₆₂₀ of 0.01 in 2 ml of sBHI. The plates were incubated at 37°C for 24 h without shaking. After incubation, the wells were washed with distilled water and stained for 15 min in the dark with the cell-permeating fluorescent dye SYTO 17 Red Fluorescent Nucleic Acid Stain in accordance with the manufacturer’s instructions (Molecular Probes).
Samples were washed three times to remove nonspecific staining, and fluorescence was observed by confocal laser microscopy. Images of the double-labeled sections were acquired with a Leica TCS-SL filter-free spectral confocal laser scanning microscope (Leica Microsystems, Inc.) with a 488-nm argon laser, 543- and 633-nm He/Ne lasers (Centres Científics i Tecnològics, Campus de Bellvitge, Universitat de Barcelona, Barcelona, Spain), a 63× oil immersion objective (1.4 numerical aperture), and an image resolution of 1,024 by 1,024 pixels. The images were acquired randomly from the cover glass surface and analyzed with the Leica Confocal Software 2.5 (Leica Microsystems, Inc.).