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The pinhole of the confocal microscope was kept at 50 μm.
In order to obtain maximum stability of the mechanical set-up, the microscope was kept working overnight before the relevant measurements were performed.
The distance from the cornea to the microscope was kept stable using a single-use contact element in sterile packaging, (TomoCap, Heidelberg Engineering, Heidelberg, Germany).
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The laser, pinhole and gain settings of the confocal microscope were kept identical among different treatments.
For semi-quantitative measurement of fluorescence intensities, laser, pinhole and gain settings of the confocal microscope were kept identical among treatments.
Since fluorescence intensity was quantified for each image, the gain and offset settings of the microscope were kept constant over the duration of each experiment.
The imaging box and microscope were kept at 36.5°C by a climate chamber that surrounds the whole stage of the microscope, including the objectives.
All optical settings of the microscope were kept constant for all image acquisitions, in order to be able to compare the acquired information.
The microscope incubation chamber temperature was kept at 37°C with a constant flux (25 l/h) of 5% CO2 humidified at 96%.
The ear was kept flat on a microscope glass.
The suspension was kept at 4°C while quantification of live and dead parasites was carried out under a light microscope.
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