Widefield ti eclipse inverted microscope

Appropriate dilutions of cultures or spore suspensions were placed on agarose pads (MOPS medium supplemented with 1.5% LSL-LE 8200 agarose [Lonza, Basel, Switzerland] and 30 mM l-valine) on a microscopy slide and covered with a cover glass attached to a 125 μL Gene Frame (Thermo Fisher Scientific, Waltham, MA, USA). The process of making agarose pads has been previously described in more detail by De Jong et al. (55 (link)). Automated TLFM monitoring was performed on a widefield Ti-Eclipse inverted microscope (Nikon, Champigny-sur-Marne, France) equipped with a 60× Plan Apo λ oil objective, a TI-CT-E motorized condenser, and a Nikon DS-Qi2 camera. GFP was imaged using a quad-edge dichroic (395/470/550/640 nm) and a FITC single emission filter. A SpectraX LED illuminator (Lumencor, Beaverton, OR, USA) was used as light source, using the 470/24 excitation filter. Temperature was controlled at 37°C with an Okolab cage incubator (Okolab, Ottaviano, Italy). While phase contrast images were taken every 15 min, GFP was imaged every 30 min in order to avoid bleaching. Images were acquired using NIS-Elements software (Nikon), and the resulting pictures were further handled with the open source software ImageJ. During acquisition of fluorescent images, photobleaching was reduced by lowering the intensity of excitation light and prolonging time intervals between exposures.

The presence and proportion of cataract lenses were quantified in coronal (optical) sections at the center of the anterior-posterior axis of 1.5–3 mpf fish. An additional 6 mpf group was analyzed by OCT for the presence of cataracts and relative changes in axial length. For this 6 mpf time-point, the extent of the opacities in the lens was quantified in ImageJ. Binary images of coronal lens sections were created and by automated thresholding, the ratio of opaque pixels was measured. For the ex vivo quantification of the proportion of cataract lenses in the 6 mpf group, a differential interference contrast microscope (Nikon WideField Ti-Eclipse inverted microscope with Coolsnap CCD camera) was used. Lenses were classified as cataractous when the nuclear ring structure was clearly visible.