Plan apo ir 60 objective

Two-photon imaging microscopy was performed to obtain 2D representations of the two Saccharomyces cerevisiae strains. The images were collected on a Nikon A1-MP scanning microscope equipped with a Plan APO IR × 60 objective (NA, 1.27; Water Immersion, Nikon, Tokyo, Japan) at a scanning speed of one frame per second. An IR laser (Chameleon, Coherent, Palo Alto, CA, USA) was used to provide excitation at 820 nm. Fluorescence emission was collected on two detection channels: FF01-492/SP (400–492 nm) and FF03-525/50 (500–550 nm) (Semrock, New York, USA). The images provided in this article were obtained by merging these two detection channels.
Samples of the culture were collected and centrifuged at 8000× g for 5 min at 20 °C. The culture supernatant was removed, and the pellet was resuspended in PBS buffer (137 mM NaCl, 2.7 mM KCl, and 11.9 mM phosphate, pH 7.2) (ThermoFisher Scientific, Illkrich, France). Five microliters of cell suspension were placed between a glass slide and a coverslip for observation.

Two-photon microscopy was performed to achieve a 3D representation of the internal structure of films with a distribution of the CLE sample through the chitosan polymer matrix. Imaging was carried out with a Plan Apo IR×60 objective (NA: 1.27, Water Immersion, Nikon, Japan) at a scanning speed of 1 frame per second. An IR laser (Chameleon, Coherent) was used to provide a 750 nm excitation. The autofluorescence emission from the CLE was collected on four detection channels (FF01-492/SP-25 (400−492 nm), FF03-525/50-25 (500−550 nm), FF01-575/25-25 (563−588 nm), and FF01-629/56-25 (601−657 nm). Increasing laser intensity was used along with the film depth for 3D images to compensate for thickness. For this method, the films were previously cast onto a coverslip and stored at 100% RH before observation [41 (link)].